Kit for multifunctional compounds forming crosslinked biomaterials

ABSTRACT

Multifunctional compounds are provided that readily crosslink in situ to provide crosslinked biomaterials. The multifunctional compounds contain a single component having at least three reactive functional groups thereon, with the functional groups selected so as to be non-reactive in an initial environment and inter-reactive in a modified environment. Reaction of a plurality of the multifunctional compounds results in a three-dimensional crosslinked matrix. In one embodiment, a first functional group is nucleophilic, a second functional group is electrophilic, and at least one additional functional group is nucleophilic or electrophilic. Methods for preparing and using the multifunctional compounds, and kits including the multifunctional compounds are also provided. Exemplary uses for the multifunctional compounds include tissue augmentation, biologically active agent delivery, bioadhesion, and prevention of adhesions following surgery or injury.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/618,989, filed Sep. 14, 2012, now allowed, which is a continuation ofU.S. patent application Ser. No. 11/575,484, filed Mar. 16, 2007, issuedas U.S. Pat. No. 8,303,973; which is a national stage application filedunder 35 U.S.C. 371 of International Application No. PCT/US2005/033367,accorded an International Filing Date of Sep. 19, 2005; which claims thebenefit of U.S. Provisional Application No. 60/611,077, filed Sep. 17,2004; these applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

This invention relates generally to multifunctional compounds, systemsfor forming crosslinked biomaterials, the crosslinked biomaterialsprepared thereby, and to methods of using such compositions. Suchmethods include the use of the crosslinked biomaterials as bioadhesivesand for tissue augmentation; for prevention of surgical adhesions; forcoating surfaces of synthetic implants; as drug delivery matrices; forophthalmic applications; and for other applications, as discussed hereinand/or as appreciated by one of ordinary skill in the art.

BACKGROUND OF THE INVENTION

Much work has been done in developing bioadhesive materials. U.S. Pat.No. 5,162,430 to Rhee et al. describes the use of collagen-syntheticpolymer conjugates prepared by covalently binding collagen to synthetichydrophilic polymers such as various derivatives of polyethylene glycol.In a related patent, U.S. Pat. No. 5,328,955 to Rhee et al., variousactivated forms of polyethylene glycol and various linkages aredescribed, which can be used to produce collagen-synthetic polymerconjugates having a range of physical and chemical properties. U.S. Pat.No. 5,324,775 to Rhee et al. also describes synthetic hydrophilicpolyethylene glycol conjugates, but the conjugates involve naturallyoccurring polymers such as polysaccharides.

EP 0 732 109 A1 to Rhee discloses a crosslinked biomaterial compositionthat is prepared using a hydrophobic crosslinking agent, or a mixture ofhydrophilic and hydrophobic crosslinking agents, where the preferredhydrophobic crosslinking agents include hydrophobic polymers thatcontain, or can be chemically derivatized to contain, two or moresuccinimidyl groups.

U.S. Pat. No. 5,580,923 to Yeung et al. discloses surgical adhesivematerial that comprises a substrate material and an anti-adhesionbinding agent. The substrate material is preferably collagen and thebinding agent preferably comprises at least one tissue-reactivefunctional group and at least one substrate-reactive functional group.

U.S. Pat. No. 5,614,587 to Rhee et al. describes bioadhesives thatcomprise collagen that is crosslinked using a multifunctionallyactivated synthetic hydrophilic polymer.

U.S. Pat. No. 5,874,500 to Rhee et al. describes a crosslinked polymercomposition that comprises one component having multiple nucleophilicgroups and another component having multiple electrophilic groups.Covalently bonding of the nucleophilic and electrophilic groups forms athree dimensional matrix that has a variety of medical uses includingtissue adhesion, surface coatings for synthetic implants, and drugdelivery. More recent developments include the addition of a thirdcomponent having either nucleophilic or electrophilic groups, as isdescribed in U.S. Pat. No. 6,458,889 to Trollsas et al.

However, in spite of the advances in the art, there remains a need forimproved crosslinked biomaterials that are easy to use and store. Thisneed, as well as others, are met by the instant invention, which is amultifunctional compound having a core substituted with at least threereactive groups, wherein each reactive group is capable of reacting withat least one other reactive group, and wherein the compound isessentially non-reactive in an initial environment. Upon reaction, athree-dimensional matrix is formed.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a multifunctional compoundcomprising a core substituted with at least three reactive groups. Thecompound is essentially non-reactive in an initial environment but isrendered reactive upon exposure to a modification in the initialenvironment that provides a modified environment such that a pluralityof the multifunctional compounds inter-react in the modified environmentto form a three-dimensional matrix. The multifunctional compound isparticularly suitable for application involving contact between abiological system and the multifunctional compound and thethree-dimensional matrix formed therefrom.

Still another aspect of the invention pertains to a multifunctionalcompound having the formula (I):

wherein n is an integer from 1-12, and when n is 2-12, each Z componentmay be different; R is selected from hydrophilic polymers, hydrophobicpolymers, amphiphilic polymers, C₂₋₁₄ hydrocarbyls, andheteroatom-containing C₂₋₁₄ hydrocarbyls; X, Y, and Z are reactivegroups and can be the same or different, each one of which is capable ofreacting with at least one other reactive group; L¹, L², and L³ arelinking groups; and p, q and r are integers from 0-1. The compound isessentially non-reactive in an initial environment but is renderedreactive upon exposure to a modification in the initial environment thatprovides a modified environment such that a plurality of themultifunctional compounds inter-react in the modified environment toform a three-dimensional matrix.

Another aspect of the invention pertains to a multifunctional compoundhaving the formula (II):[X′-(L⁴)_(a)-Y′-(L⁵)_(b)]_(c)-R′  (II)

wherein a and b are integers from 0-1; c is an integer from 3-12; R′ isselected from hydrophilic polymers, hydrophobic polymers, amphiphilicpolymers, C₂₋₁₄ hydrocarbyls, and heteroatom-containing C₂₋₁₄hydrocarbyls; X′ and Y′ are reactive groups and can be the same ordifferent; and L⁴ and L⁵ are linking groups.

In a preferred embodiment of formula (I), X is a nucleophilic group, Y,is an electrophilic group, and Z is an electrophilic or a nucleophilicgroup.

In a preferred embodiment of formula (II), X′ is a nucleophilic groupand Y′ is an electrophilic group.

With formulas (I) and (II), the reactive groups may be selected fromnucleophilic groups, electrophilic groups, redox groups, oxidativecoupling reactive groups, photoinitiated reactive groups, andtemperature-sensitive groups. In one embodiment, the reactive groups arenucleophilic and electrophilic groups that undergo a nucleophilicsubstitution reaction, a nucleophilic addition reaction, or both. Thenucleophilic groups may be selected from —NH₂, —NHR′, —N(R¹)₂, —SH, —OH,—COOH, —C₆H₄—OH, —H, —PH₂, —PHR′, —P(R¹)₂, —NH—NH₂, —CO—NH—NH₂, and—C₅H₄N, where R¹ is a hydrocarbyl group, and each R¹ may be the same ordifferent. The electrophilic groups may be selected from —CO—Cl,—(CO)—O—(CO)—R (where R is an alkyl group), —CH═CH—CH═O and—CH═CH—C(CH₃)═O, halo, —N═C═O, —N═C═S, —SO₂CH═CH₂, —O(CO)—C═CH₂,—O(CO)—C(CH₃)═CH₂, —S—S—(C₅H₄N), —O(CO)—C(CH₂CH₃)═CH₂, —CH═CH—C═NH,—COOH, —(CO)O—N(COCH₂)₂, —CHO, —(CO)O—N(COCH₂)₂—S(O)₂OH, and —N(COCH)₂.

In one embodiment of formulas (I) and (II), the nucleophilic groups areamino groups and the electrophilic groups are amine-reactive groups. Theamine-reactive groups may contain an electrophilically reactive carbonylgroup susceptible to nucleophilic attack by a primary or secondaryamine. The amine-reactive groups may be selected from carboxylic acidesters, acid chloride groups, anhydrides, ketones, aldehydes, halo,isocyanato, thioisocyanato, epoxides, activated hydroxyl groups,olefins, carboxyl, succinimidyl ester, sulfosuccinimidyl ester,maleimido, epoxy, and ethenesulfonyl.

In another embodiment of formulas (I) and (II), the nucleophilic groupsare sulfhydryl groups and the electrophilic groups aresulfhydryl-reactive groups. The sulfhydryl-reactive groups may beselected from mixed anhydrides; ester derivatives of phosphorus; esterderivatives of p-nitrophenol, p-nitrothiophenol and pentafluorophenol;esters of substituted hydroxylamines, including N-hydroxyphthalimideesters, N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters,and N-hydroxyglutarimide esters; esters of 1-hydroxybenzotriazole;3-hydroxy-3,4-dihydro-benzotriazin-4-one;3-hydroxy-3,4-dihydro-quinazoline-4-one; carbonylimidazole derivatives;acid chlorides; ketenes; and isocyanates. The sulfhydryl-reactive groupsmay be selected so as to form a thioester, imido-thioester, thioether,or disulfide linkage upon reaction with the sulfhydryl groups. Where thesulfhydryl-reactive groups form a disulfide linkage, they may have thestructure —S—S—Ar where Ar is a substituted or unsubstitutednitrogen-containing heteroaromatic moiety or a non-heterocyclic aromaticgroup substituted with an electron-withdrawing moiety. Where thesulfhydryl-reactive groups form a thioether linkage, they may beselected from maleimido, substituted maleimido, haloalkyl, epoxy, imino,aziridino, olefins, and α,β-unsaturated aldehydes and ketones.

In another embodiment of formulas (I) and (II), the reactive groupsundergo an oxidation-reduction reaction and are vinyl groups.

In a further embodiment of formulas (I) and (II), the reactive groupsare oxidative coupling reactive groups and are halo groups, with anadjacent electron-withdrawing group on the halogen-bearing carbon.

In still another embodiment of formulas (I) and (II), the reactivegroups are photoinitiated reactive groups and are selected from azide,alkyl, and benzophenone.

In yet another embodiment of formulas (I) and (II), the reactive groupsare temperature sensitive groups and are vinyl groups.

In the embodiment of formulas (I) and (II), where R is a hydrophilicpolymer, the hydrophilic polymer may be linear, branched, dendrimeric,hyperbranched, or star polymer.

In a preferred embodiment, the R group of formulas (I) and (II) is ahydrophilic polymer selected from polyalkylene oxides; polyols;poly(oxyalkylene)-substituted diols and polyols; polyoxyethylatedsorbitol; polyoxyethylated glucose; poly(acrylic acids) and analogs andcopolymers thereof; polymaleic acids; polyacrylamides; poly(olefinicalcohols); poly(N-vinyl lactams); polyoxazolines; polyvinylamines; andcopolymers thereof. The polyalkylene oxide or polyol may be selectedfrom polyethylene glycol and poly(ethylene oxide)-poly(propylene oxide)copolymers. The polyol may be selected from glycerol, polyglycerol, andpropylene glycol. The poly(oxyalkylene)-substituted polyol may beselected from mono-, di- and tri-polyoxyethylated glycerol, mono- anddi-polyoxyethylated propylene glycol, and mono- and di-polyoxyethylatedtrimethylene glycol. The poly(acrylic acid), analog or copolymer thereofmay be selected from poly(acrylic acid), poly(methacrylic acid),poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide acrylates), and poly(methylalkylsulfoxidemethacrylates). The polyacrylamide may be selected from polyacrylamide,poly(methacrylamide), poly(dimethylacrylamide),poly(N-isopropylacrylamide), and copolymers thereof. The poly(olefinicalcohol) may be selected from poly (vinyl alcohols) and copolymersthereof. The poly(N-vinyl lactam) may be selected from poly(vinylpyrrolidones), poly(vinyl caprolactams), and copolymers thereof. Thepolyoxazoline may be selected from poly(methyloxazoline) andpoly(ethyloxazoline).

In another embodiment of formulas (I) and (II), the R group is ahydrophilic polymer selected from proteins, carboxylatedpolysaccharides, aminated polysaccharides, and activatedpolysaccharides. In a preferred embodiment, the hydrophilic polymer isselected from collagen and glycosaminoglycans.

Where the R group of formulas (I) and (II) is a hydrophobic polymer, thehydrophobic polymer may contain repeating monomer units. In a preferredembodiment, the hydrophobic polymer selected from polylactic acid andpolyglycolic acid.

Where the R group of formulas (I) and (II) is an amphiphilic polymer,the amphiphilic polymer may contain repeating monomer units.

Where the R group of formulas (I) and (II) is a C₂₋₁₄ hydrocarbyl, theC₂₋₁₄ hydrocarbyl may be selected from alkanes, diols, polyols, andpolyacids.

Where the R group of formulas (I) and (II) is a heteroatom-containingC₂₋₁₄ hydrocarbyl, heteroatom-containing C₂₋₁₄ hydrocarbyl may beselected from di- and poly-electrophiles.

In another embodiment of formulas (I) and (II), the inter-reactioncomprises formation of covalent bonds, noncovalent bonds, or both. Thenoncovalent bonds may be ionic bonds, hydrogen bonds, or association ofhydrophobic molecular segments. In a preferred embodiment, all of themolecular segments are the same.

In a further embodiment of formulas (I) and (II), the linking groupsprovide hydrolyzable linkages selected from ester linkages, anhydridelinkages, ortho ester linkages, ortho carbonate linkages, amidelinkages, phosphoester linkages, alpha-hydroxy acid linkages,lactone-based linkages, and amide linkages. In another embodiment, thelinking groups provide non-degradable linkages selected fromsuccinimide, propionic acid, and carboxymethylate linkages. In stillanother embodiment, the linking groups provide enzymatically degradablelinkages selected from Leu-Gly-Pro-Ala, which is degraded bycollagenase, and Gly-Pro-Lys, which is degraded by plasmin.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising the multifunctional compound. A pharmaceutically acceptablecarrier may also be included.

The multifunctional compound may further comprise a biologically activeagent with or without a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier may be a micelle, a microsphere, ora nanosphere.

Where the pharmaceutically acceptable carrier is a microsphere or ananosphere, the pharmaceutically acceptable carrier may be a degradablepolymer, such as a polyester, and the polyester may be aglycolide/lactide copolymer. The degradable polymer may also becomprised of residues of one or more monomers selected from the groupconsisting of lactide, lactic acid, glycolide, glycolic acid,ε-caprolactone, γ-caprolactone, hydroxyvaleric acid, hydroxybutyricacid, β-butyrolactone, γ-butyrolactone, γ-valerolactone,γ-decanolactone, δ-decanolactone, trimethylene carbonate,1,4-dioxane-2-one or 1,5-dioxepan-2one).

The multifunctional compound may further comprise a biologically activeagent.

In one embodiment of the invention, the multifunctional compound furthercomprises a biologically active agent that is an anti-fibrotic agent. Asused in the multifunctional compound, the anti-fibrotic agent may beused to inhibit any of the following: cell regeneration, angiogenesis,fibroblast migration, fibroblast proliferation, deposition ofextracellular matrix, tissue remodeling, adenosine deaminase, purinering synthesis, dihydrofolate reduction, ribonucleotide synthesis orfunction, thymidine monophosphate synthesis or function, DNA synthesis,protein synthesis, and microtubule function. The anti-fibrotic agent mayalso be used to block thymidine monophosphate, to cause DNA damage, andto cause DNA adduct formation.

Any of the following anti-fibrotic agents may be used in themultifunctional compound: an angiogenesis inhibitor; a 5-lipoxygenaseinhibitor or antagonist; a chemokine receptor antagonist; a cell cycleinhibitor; a taxane; an anti-microtubule agent; paclitaxel; an analogueor derivative of paclitaxel; a vinca alkaloid; camptothecin or ananalogue or derivative thereof; a podophyllotoxin, wherein thepodophyllotoxin may be an etoposide or an analogue or derivativethereof; an anthracycline, wherein the anthracycline may be doxorubicinor an analogue or derivative thereof or the anthracycline may bemitoxantrone or an analogue or derivative thereof; a platinum compound;a nitrosourea; a nitroimidazole; a folic acid antagonist; a cytidineanalogue; a pyrimidine analogue; a fluoropyrimidine analogue; a purineanalogue; a nitrogen mustard or an analogue or derivative thereof; ahydroxyurea; a mytomicin or an analogue or derivative thereof; an alkylsulfonate; a benzamide or an analogue or derivative thereof; anicotinamide or an analogue or derivative thereof; a halogenated sugaror an analogue or derivative thereof; a DNA alkylating agent; ananti-microtubule agent; a topoisomerase inhibitor; a DNA cleaving agent;an antimetabolite; a nucleotide interconversion inhibitor; ahydroorotate dehydrogenase inhibitor; a DNA intercalation agent; an RNAsynthesis inhibitor; a pyrimidine synthesis inhibitor; a cyclindependent protein kinase inhibitor; an epidermal growth factor kinaseinhibitor; an elastase inhibitor; a factor Xa inhibitor; afarnesyltransferase inhibitor; a fibrinogen antagonist; a guanylatecyclase stimulant; a heat shock protein 90 antagonist; which may be ageldanamycin or an analogue or derivative thereof; a guanylate cyclasestimulant; a HMGCoA reductase inhibitor, which may be simvastatin or ananalogue or derivative thereof; an IKK2 inhibitor; an IL-1 antagonist;an ICE antagonist; an IRAK antagonist; an IL-4 agonist; animmunomodulatory agent; sirolimus or an analogue or derivative thereof;everolimus or an analogue or derivative thereof; tacrolimus or ananalogue or derivative thereof; biolmus or an analogue or derivativethereof; tresperimus or an analogue or derivative thereof; auranofin oran analogue or derivative thereof; 27-0-demethylrapamycin or an analogueor derivative thereof; gusperimus or an analogue or derivative thereof;pimecrolimus or an analogue or derivative thereof; ABT-578 or ananalogue or derivative thereof; an inosine monophosphate dehydrogenase(IMPDH) inhibitor, which may be mycophenolic acid or an analogue orderivative thereof or 1-α-25 dihydroxy vitamin D₃ or an analogue orderivative thereof; a leukotriene inhibitor; an MCP-1 antagonist; an MMPinhibitor; an NF kappa B inhibitor, which may be Bay 11-7082; an NOantagonist; a p38 MAP kinase inhibitor, which may be SB 202190; aphosphodiesterase inhibitor; a TGF-β inhibitor; a thromboxane A2antagonist; a TNF-α antagonist; a TACE inhibitor; a tyrosine kinaseinhibitor; vitronectin inhibitor; a fibroblast growth factor inhibitor;a protein kinase inhibitor; a PDGF receptor kinase inhibitor; anendothelial growth factor receptor kinase inhibitor; a retinoic acidreceptor antagonist; a platelet derived growth factor receptor kinaseinhibitor; a fibrinogen antagonist; an antimycotic agent; sulconizole; abisphosphonate; a phospholipase A1 inhibitor; a histamine H1/H2/H3receptor antagonist; a macrolide antibiotic; a GPIIb/IIIa receptorantagonist; an endothelin receptor antagonist; a peroxisomeproliferator-activated receptor agonist; an estrogen receptor agent; asomastostatin analogue; a neurokinin 1 antagonist; a neurokinin 3antagonist; a VLA-4 antagonist; an osteoclast inhibitor; a DNAtopoisomerase ATP hydrolyzing inhibitor; an angiotensin I convertingenzyme inhibitor; an angiotensin II antagonist; an enkephalinaseinhibitor; a peroxisome proliferator-activated receptor gamma agonistinsulin sensitizer; a protein kinase C inhibitor; a ROCK (rho-associatedkinase) inhibitor; a CXCR3 inhibitor; Itk inhibitor; a cytosolicphospholipase A₂-α inhibitor; a PPAR agonist; an immunosuppressant; anErb inhibitor; an apoptosis agonist; a lipocortin agonist; a VCAM-1antagonist; a collagen antagonist; an α-2 integrin antagonist; a TNF-αinhibitor; a nitric oxide inhibitor; and a cathepsin inhibitor.

In another embodiment of the invention, the multifunctional compoundfurther comprises a biologically active agent that is a fibrosing agent.As used in the multifunctional compound, the anti-fibrotic agent may beused to promote any of the following; regeneration; angiogenesis;fibroblast migration; fibroblast proliferation; deposition ofextracellular matrix (ECM); and tissue remodeling. The fibrosing agentmay also be used as an arterial vessel wall irritant.

Fibrosing agents that may be used in the multifunctional compound may beor may be comprised of silk; silkworm silk; spider silk; recombinantsilk; raw silk; hydrolyzed silk; acid-treated silk; acylated silk;mineral particles; talc; chitosan; polylysine; fibronectin; bleomycin;or CTGF. The fibrosing agent may also be in the form of a particulate,which may be a biodegradable particulate or a non-biodegradableparticulate. Biodegradable particulates may be comprised of a materialselected from the group consisting of polyester, polyanhydride,poly(anhydride ester), poly(ester-amide), poly(ester-urea),polyorthoester, polyphosphoester, polyphosphazine, polycyanoacrylate,collagen, chitosan, hyaluronic acid, chromic cat gut, alginate, starch,cellulose and cellulose ester. Non-biodegradable particulates may becomprised of a material selected from the group consisting of polyester,polyurethane, silicone, polyethylene, polypropylene, polystyrene,polyacrylate, polymethacrylate, and silk. Examples of preferredparticulates may be a particulate form of a member selected from thegroup consisting of silk, talc, starch, glass, silicate, silica, calciumphosphate, calcium sulfate, calcium carbonate, hydroxyapatite, syntheticmineral, polymethylmethacrylate, silver nitrate, ceramic, and otherinorganic particles.

In a further embodiment of the multifunctional compound, thebiologically active agent promotes bone growth. Within this embodiment,the fibrosing agent may promote the bone growth. Fibrosing agents thatmay promote bone growth may include a bone morphogenic protein and anosteogenic growth factor, the latter which may be selected fromtransforming growth factor, platelet-derived growth factor, andfibroblast growth factor.

In another embodiment of the invention, the multifunctional compoundwith a fibrosing agent further comprises a pharmaceutical agent thatinduces sclerosis (a sclerosant), wherein the sclerosant may be asurfactant or it may be selected from the group consisting of ethanol,dimethyl sulfoxide, sucrose, sodium chloride, dextrose, glycerin,minocycline, tetracycline, doxycycline, polidocanol, sodium tetradecylsulfate, sodium morrhuate, and sotradecol.

In a further embodiment of the invention, the multifunctional compoundwith a fibrosing agent further comprises an inflammatory cytokine, whichmay be selected from the group consisting of TGFβ, PDGF, VEGF, bFGF,TNFα, NGF, GM-CSF, IGF-a, IL-1, IL-1-β, IL-8, IL-6, and growth hormone.

In still another embodiment of the invention, the multifunctionalcompound with a fibrosing agent further comprises an agent thatstimulates cell proliferation, which may be selected from the groupconsisting of dexamethasone, isotretinoin (13-cis retinoic acid),17-β-estradiol, estradiol, 1-α-25 dihydroxyvitamin D₃,diethylstibesterol, cyclosporine A, L-NAME, all-trans retinoic acid(ATRA), and analogues and derivatives thereof.

In a further embodiment of the multifunctional compound, thebiologically active agent is mixed with the multifunctional compound toform a mixture.

In another embodiment of the multifunctional compound, the biologicallyactive agent is chemically coupled to the multifunctional compound.

Yet another aspect of the invention relates to a method of forming athree-dimensional matrix comprising the steps of: (a) providing aplurality of multifunctional compounds each comprising a coresubstituted with at least three reactive groups, as described above, and(b) activating the plurality of multifunctional compounds to effectinter-reaction in the modified environment to form a three-dimensionalmatrix.

Still another aspect of the invention pertains to a method of adheringtissue of a patient comprising the steps of: (a) placing into contactwith tissue an adhesive composition comprising a plurality ofmultifunctional compounds, as described above, and (b) activating theplurality of multifunctional compounds to effect inter-reaction in themodified environment to form a three-dimensional matrix to adhere thetissue.

Another aspect of the invention relates to a method of forming athree-dimensional matrix comprising the steps of: (a) providing amultifunctional compound of the invention; and (b) rendering thenucleophilic and electrophilic groups reactive by exposing thecomposition to an aqueous environment to effect inter-reaction; whereinsaid exposure comprises: (i) dissolving the composition in a firstbuffer solution having a pH within the range of about 1.0 to 5.5 to forma homogeneous solution, and (ii) adding a second buffer solution havinga pH within the range of about 6.0 to 11.0 to the homogeneous solution;and (c) allowing a three-dimensional matrix to form. A preferredcomposition for use in this method is the multifunctional compound. Thethree-dimensional matrix of the invention may be formed without input ofany external energy or by polymerization.

In a preferred embodiment, the pH of the first buffer solution isselected to retard the reactivity of the nucleophilic groups on themultifunctional compound by rendering the nucleophilic groups relativelynon-nucleophilic. In this preferred embodiment, the second buffersolution neutralizes the effect of the first buffer solution, so thatthe nucleophilic groups of the multifunctional compound regain theirnucleophilic character and inter-react with the electrophilic groups ofthe second component.

In another preferred embodiment, the multifunctional compound, firstbuffer solution and second buffer solution are housed separately in amultiple-compartment syringe system having a multiple barrels, a mixinghead, and an exit orifice; step (b)(i) comprises adding the first buffersolution to the barrel housing the multifunctional compound to dissolvethe composition and form a homogeneous solution, and extruding thehomogeneous solution into the mixing head; step (b)(ii) comprisessimultaneously extruding the second buffer solution into the mixinghead; and step (c) further comprises extruding the resulting compositionthrough the orifice onto a surface.

Yet another aspect of the invention relates to a method of sealingtissue of a patient comprising the steps of: (a) providing a pluralityof a multifunctional compound of the invention; (b) rendering thenucleophilic and electrophilic groups reactive by exposing themultifunctional compound to an aqueous environment to effectinter-reaction; wherein said exposure comprises: (i) dissolving themultifunctional compound in a first buffer solution having a pH withinthe range of about 1.0 to 5.5 to form a homogeneous solution, and (ii)adding a second buffer solution having a pH within the range of about6.0 to 11.0 to the homogeneous solution to form a mixture; and (c)placing the mixture into contact with tissue and allowing athree-dimensional matrix to form and seal the tissue. A preferredcomposition for use in this method is the multifunctional compound.

Still another aspect of the invention relates to a method of preventingadhesions between tissues of a patient comprising the steps of: (a)providing a plurality of a multifunctional compound of the invention;(b) rendering the nucleophilic and electrophilic groups reactive byexposing the multifunctional compound to an aqueous environment toeffect inter-reaction; wherein said exposure comprises: (i) dissolvingthe multifunctional compound in a first buffer solution having a pHwithin the range of about 1.0 to 5.5 to form a homogeneous solution, and(ii) adding a second buffer solution having a pH within the range ofabout 6.0 to 11.0 to the homogeneous solution to form a mixture; and (c)placing the mixture into contact with tissue and allowing athree-dimensional matrix to form on the tissue. A preferred compositionfor use in this method is the multifunctional compound.

A further aspect of the invention relates to a method of forming athree-dimensional matrix on a surface of a device comprising the stepsof: (a) providing a plurality of a multifunctional compound of theinvention; and (b) rendering the nucleophilic and electrophilic groupsreactive by exposing the multifunctional compound to an aqueousenvironment to effect inter-reaction; wherein said exposure comprises:(i) dissolving the multifunctional compound in a first buffer solutionhaving a pH within the range of about 1.0 to 5.5 to form a homogeneoussolution, and (ii) adding a second buffer solution having a pH withinthe range of about 6.0 to 11.0 to the homogeneous solution; and (c)applying the homogeneous solution to a surface of a device and allowingthe three-dimensional matrix to form. A preferred composition for use inthis method is the multifunctional compound.

Another aspect of the invention relates to a method of preventingscarring in the vicinity of a medical implant comprising the steps of:(a) providing a plurality of a multifunctional compound of theinvention, wherein the multifunctional compound further comprises ananti-fibrotic agent; (b) rendering the nucleophilic and electrophilicgroups reactive by exposing the multifunctional compound to an aqueousenvironment to effect inter-reaction; wherein said exposure comprises:(i) dissolving the multifunctional compound in a first buffer solutionhaving a pH within the range of about 1.0 to 5.5 to form a homogeneoussolution, and (ii) adding a second buffer solution having a pH withinthe range of about 6.0 to 11.0 to the homogeneous solution to form amixture; (c) applying the mixture to a surface of a medical implant andallowing a three-dimensional matrix to form on the surface of themedical implant; and (d) placing the medical implant into an animalhost, wherein release of the anti-fibrotic agent from the compositioninhibits scarring in the animal host. In one embodiment, theanti-fibrotic agent is released into tissue in the vicinity of theimplant after deployment of the implant. A preferred composition for usein this method is the multifunctional compound with an anti-fibroticagent.

Yet another aspect of the invention relates to a method of promotingscarring in the vicinity of a medical implant comprising the steps of:(a) providing a plurality of a multifunctional compound of theinvention, where the multifunctional compound further comprises afibrosing agent; (b) rendering the nucleophilic and electrophilic groupsreactive by exposing the multifunctional compound to an aqueousenvironment to effect inter-reaction; wherein said exposure comprises:(i) dissolving the multifunctional compound in a first buffer solutionhaving a pH within the range of about 1.0 to 5.5 to form a homogeneoussolution, and (ii) adding a second buffer solution having a pH withinthe range of about 6.0 to 11.0 to the homogeneous solution; and (c)applying the mixture to a surface of a medical implant and allowing athree-dimensional matrix to form on the surface of the medical implant;and (d) placing the medical implant into an animal host, wherein releaseof the fibrotic agent from the matrix inhibits scarring in the animalhost. In a preferred embodiment, the fibrosing agent is released intotissue in the vicinity of the implant after deployment of the implant. Apreferred composition for use in this method is the multifunctionalcompound with a fibrosing agent.

Still another aspect of the invention relates to a kit for use in foruse in medical applications, comprising: (a) a plurality of amultifunctional compound of the invention; (b) a first buffer solutionhaving a pH within the range of about 1.0 to 5.5; and (c) a secondbuffer solution having a pH within the range of about 6.0 to 11.0,wherein the nucleophilic and electrophilic groups are non-reactive in adry environment but are rendered reactive upon exposure to an aqueousenvironment such that the plurality of multifunctional compoundsinter-react in the aqueous environment to form a three-dimensionalmatrix and further wherein each component is packaged separately andadmixed immediately prior to use.

Another aspect of the invention relates to a kit for use in medicalapplications, comprising: (a) a plurality of a multifunctional compoundof the invention; (b) a first buffer solution having a pH within therange of about 1.0 to 5.5; and (c) a second buffer solution having a pHwithin the range of about 6.0 to 11.0, wherein each component ispackaged separately and admixed immediately prior to use. A preferredcomposition of the invention for use in this kit is the multifunctionalcompound. It is preferred that each component of the kit is in aseparate sterile package.

The kit may further comprise a delivery device, which in one embodiment,may be a multi-compartment device. A preferred multi-compartment deviceof the invention is a multiple-compartment syringe system havingmultiple barrels, a mixing head, and an exit orifice. Where the kit is amultiple-compartment syringe system, the multifunctional compound, thefirst buffer solution, and the second buffer solution are housedseparately in the multiple-compartment syringe system.

In another embodiment of the invention, the delivery device is apressurized delivery system. A preferred pressurized delivery systemcomprises: a plurality of fluid component inlets each adapted tocommunicate with a source of different fluid components; at least onecarrier fluid inlet adapted to communicate with a source of apressurized carrier fluid; a diffuser surface located downstream fromthe plurality of fluid component inlets and the at least one carrierfluid inlet; and an outlet extending through the diffuser surface,wherein the diffuser surface is adapted to receive fluid componentsthereon and has a shape effective to direct and maintain each receivedfluid component in a different flow path toward the outlet for mixingand dispensing therethrough by the pressurized carrier fluid from the atleast one carrier fluid inlet. Within this embodiment, a preferredpressurized carrier fluid is pressurized air and the preferred fluidcomponents are the first buffer solution and the second buffer solutionof the invention.

Another embodiment of the kit for use in medical applications furthercomprises a biologically active agent and the medical applicationinvolves delivering the biologically active agent. The biologicallyactive agent may be packaged with the multifunctional compound and mayfurther comprise a pharmaceutically acceptable carrier packaged with thebiologically active agent and the multifunctional compound. Thebiologically active agent may also be packaged as a solution with thefirst buffer or as a solution with the second buffer. The kit mayfurther comprise a pharmaceutically acceptable carrier as a fourthcomponent. The biologically active agent is packaged with thepharmaceutically acceptable carrier.

Yet another embodiment of the kit for use in medical applicationsfurther comprises living cells or genes, and the medical applicationinvolves delivering the living cells or genes.

Other medical applications that the kit may be used for include adheringor sealing biological tissue, bioadhesion, ophthalmic applications,tissue augmentation, adhesion prevention, forming a synthetic implant orcoating a synthetic implant, treatment of aneurysms, and laparoscopicprocedures.

These and other aspects of the present invention are described in detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a preferred multi-compartment syringe device of thepresent invention.

FIGS. 2 and 3 schematically illustrate an embodiment of the pressurizeddelivery device of the present invention that includes a cap having aninterior diffuser surface and a lumen assembly for delivering fluidcomponents and a pressurized carrier fluid to the diffuser surface. FIG.1 depicts the device in exploded view and FIG. 2 depicts the interiordiffuser surface of the cap.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Nomenclature

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto particular compositional forms, crosslinkable components,crosslinking techniques, or methods of use, as such may vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example, “amultifunctional compound” refers not only to a single multifunctionalcompound but also to a combination of two or more of the same ordifferent multifunctional compounds, “a reactive group” refers to acombination of reactive groups as well as to a single reactive group,and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Although any methods and materialssimilar or equivalent to those described herein may be useful in thepractice or testing of the present invention, preferred methods andmaterials are described below. All patents, patent applications andother publications mentioned herein are incorporated herein byreference. Specific terminology of particular importance to thedescription of the present invention is defined below.

The term “inter-react” and “inter-reaction” as used herein refers to theformulation of covalent bonds, noncovalent bonds, or both. The term thusincludes crosslinking, which involves both intermolecular crosslinks andoptionally intramolecular crosslinks as well, arising from the formationof covalent bonds. Covalent bonding between two reactive groups may bedirect, in which case an atom in reactive group is directly bound to anatom in the other reactive group, or it may be indirect, through alinking group. Noncovalent bonds include ionic (electrostatic) bonds,hydrogen bonds, or the association of hydrophobic molecular segments,which may be the same or different. A crosslinked matrix may, inaddition to covalent bonds, also include such intermolecular and/orintramolecular noncovalent bonds.

When referring to polymers, the terms “hydrophilic” and “hydrophobic”are generally defined in terms of an HLB value, i.e., a hydrophiliclipophilic balance. A high HLB value indicates a hydrophilic compound,while a low HLB value characterizes a hydrophobic compound. HLB valuesare well known in the art, and generally range from 1 to 18. Preferredmultifunctional compound cores are hydrophilic, although as long as themultifunctional compound as a whole contains at least one hydrophiliccomponent, crosslinkable hydrophobic components may also be present.

The term “polymer” is used not only in the conventional sense to referto molecules composed of repeating monomer units, includinghomopolymers, block copolymers, random copolymers, and graft copolymers,but also refers to polyfunctional small molecules that do not containrepeating monomer units but are “polymeric” in the sense of being“polyfunctional,” i.e., containing two or more functional groups.Accordingly, it will be appreciated that when the term “polymer” isused, difunctional and polyfunctional small molecules are included. Suchmoieties include, by way of example: the difunctional electrophilesdisuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidylpropionate) (DSP),bis(2-succinimidooxy-carbonyloxy) ethyl sulfone (BSOCOES),3,3′-dithiobis(sulfosuccinimidylpropionate (DTSSP); and the di- andpolyfunctional nucleophiles ethylenediamine (H₂N—CH₂—CH₂—NH₂),tetramethylene diamine (H₂N—[CH₂]₄—NH₂), pentamethylene diamine(cadaverine) (H₂N—[CH₂]₅—NH₂), hexamethylene diamine (H₂N—[CH₂]₆—NH₂),bos(2-aminoethyl)amine (HN—[CH₂—CH₂—NH₂]₂), and tris (2-aminoethyl)amine(N—[CH₂—CH₂—NH₂]₃). All suitable polymers herein are nontoxic,non-inflammatory, and nonimmunogenic, and will preferably be essentiallynondegradable in vivo over a period of at least several months.

The term “synthetic” is used to refer to polymers, compounds and othersuch materials that are “chemically synthesized.” For example, asynthetic material in the present compositions may have a molecularstructure that is identical to a naturally occurring material, but thematerial per se, as incorporated in the compositions of the invention,has been chemically synthesized in the laboratory or industrially.“Synthetic” materials also include semi-synthetic materials, i.e.,naturally occurring materials, obtained from a natural source, that havebeen chemically modified in some way. Generally, however, the syntheticmaterials herein are purely synthetic, i.e., they are neithersemi-synthetic nor have a structure that is identical to that of anaturally occurring material.

The term “effective amount” refers to the amount of composition requiredin order to obtain the effect desired. For example, a “tissuegrowth-promoting amount” of a composition refers to the amount needed inorder to stimulate tissue growth to a detectable degree. Tissue, in thiscontext, includes connective tissue, bone, cartilage, epidermis anddermis, blood, and other tissues. The actual amount that is determinedto be an effective amount will vary depending on factors such as thesize, condition, sex, and age of the patient and can be more readilydetermined by the caregiver.

The term “in situ” as used herein means at the site of administration.Thus, compositions of the invention can be injected or otherwise appliedto a specific site within a patient's body, e.g., a site in need ofaugmentation, and allowed to crosslink at the site of injection.Suitable sites will generally be intradermal or subcutaneous regions foraugmenting dermal support, at a bone fracture site for bone repair,within sphincter tissue for sphincter augmentation (e.g., forrestoration of continence), within a wound or suture to promote tissueregrowth, and within or adjacent to vessel anastomoses to promote vesselregrowth.

The term “aqueous medium” includes solutions, suspensions, dispersions,colloids, and the like containing water. The term “aqueous environment”means an environment containing an aqueous medium. Similarly, the term“thy environment” means an environment that does not contain an aqueousmedium.

The term “biologically active agent” refers to an organic molecule thatexerts biological effects in vivo. Examples of biologically activeagents include, by way of example and not limitation, enzymes, receptorantagonists or agonists, hormones, growth factors, autogenous bonemarrow, antibiotics, antimicrobial agents, and antibodies. The term“biologically active agent” is also intended to encompass various celltypes and genes that can be incorporated into the compositions of theinvention.

The terms “active agent,” “biologically active agent,” and “drug” areused interchangeably herein to refer to a chemical material or compoundsuitable for administration to a patient and that induces a desiredeffect. The terms include agents that are therapeutically effective aswell as prophylactically effective. Also included are derivatives andanalogs of those compounds or classes of compounds specificallymentioned that also induce the desired effect.

As used herein the terms “active agent,” “biologically active agent,”“therapeutic agent,” “pharmacologically active agent,” and “drug” referto an organic molecule that exerts biological effects in vivo. Forpurposes of this discussion, the term “biologically active agent” isused, with the understanding that the use of this term does not excludethe application to the remaining terms. Examples of biologically activeagents include, by way of example and not limitation, enzymes, receptorantagonists or agonists, hormones, growth factors, autogenous bonemarrow, antibiotics, antimicrobial agents and antibodies. The termbiologically active agent is also intended to encompass various celltypes and genes that can be incorporated into the compositions of theinvention. Other examples of biologically active agents include thosethat inhibit fibrosis and those that promote fibrosis. In certainembodiments, a biologically active agent may promote adhesion between atissue and a substrate (e.g., a surface of a medical device).

“Fibrosis,” “scarring,” or “fibrotic response” refers to the formationof fibrous tissue in response to injury or medical intervention.Therapeutic agents which promote (also referred to interchangeablyherein as “induce,” “stimulate,” “cause,” and the like) fibrosis orscarring are referred to interchangeably herein as “fibrosis-inducingagents,” “scarring agents,” “fibrosing agents,” “adhesion-inducingagents,” and the like, where these agents do so through one or moremechanisms including: inducing or promoting angiogenesis, stimulatingmigration or proliferation of connective tissue cells (such asfibroblasts, smooth muscle cells, vascular smooth muscle cells),inducing ECM production, and/or promoting tissue remodeling. Therapeuticagents which inhibit fibrosis or scarring are referred to herein as“fibrosis-inhibiting agents,” “anti-scarring agents,” and the like,where these agents inhibit fibrosis through one or more mechanismsincluding: inhibiting angiogenesis, inhibiting migration orproliferation of connective tissue cells (such as fibroblasts, smoothmuscle cells, vascular smooth muscle cells), reducing ECM production,and/or inhibiting tissue remodeling.

“Sclerosing” refers to a tissue reaction in which an irritant is appliedlocally to a tissue which results in an inflammatory reaction and isfollowed by scar tissue formation at the site of irritation. Apharmaceutical agent that induces or promotes sclerosis is referred toas a “sclerosant,” or a “sclerosing agent.” Representative examples ofsclerosants include ethanol, dimethyl sulfoxide, surfactants (e.g.,TRITON X, sorbitan monolaurate, sorbitan sesquioleate, glycerolmonostearate, and polyoxyethylene, polyoxyethylene cetyl ether, and thelike), sucrose, sodium chloride, dextrose, glycerin, minocycline,tetracycline, doxycycline, polidocanol, sodium tetradecyl sulfate,sodium morrhuate, ethanolamine, phenol, sarapin and sotradecol.

“Anti-microtubule agents” should be understood to include any protein,peptide, chemical, or other molecule which impairs the function ofmicrotubules, for example, through the prevention or stabilization ofpolymerization. Compounds that stabilize polymerization of microtubulesare referred to herein as “microtubule stabilizing agents.” A widevariety of methods may be utilized to determine the anti-microtubuleactivity of a particular compound, including for example, assaysdescribed by Smith et al. (Cancer Lett 79(2):213-219 (1994)) andMooberry et al., (Cancer Lett. 96(2):261-266 (1995)). The terms “medicaldevice,” “implant,” “medical implant,” and the like are usedsynonymously to refer to any object that is designed to be placedpartially or wholly within a patient's body for one or more therapeuticor prophylactic purposes such as for restoring physiological function,alleviating symptoms associated with disease, delivering therapeuticagents, and/or repairing or replacing or augmenting damaged or diseasedorgans and tissues. While normally composed of biologically compatiblesynthetic materials (e.g., medical-grade stainless steel, titanium andother metals; polymers such as polyurethane, silicon, PLA, PLGA andother materials) that are exogenous, some medical devices and implantsinclude materials derived from animals (e.g., “xenografts” such as wholeanimal organs; animal tissues such as heart valves; naturally occurringor chemically-modified molecules such as collagen, hyaluronic acid,proteins, carbohydrates and others), human donors (e.g., “allografts”such as whole organs; tissues such as bone grafts, skin grafts andothers), or from the patients themselves (e.g., “autografts” such assaphenous vein grafts, skin grafts, tendon/ligament/muscle transplants).

With regard to nomenclature pertinent to molecular structures, thefollowing definitions apply:

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group typically although not necessarilycontaining 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, aswell as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.Generally, although again not necessarily, alkyl groups herein contain 1to about 12 carbon atoms. The term “lower alkyl” intends an alkyl groupof one to six carbon atoms, preferably one to four carbon atoms.“Substituted alkyl” refers to alkyl substituted with one or moresubstituent groups. “Alkylene,” “lower alkylene” and “substitutedalkylene” refer to divalent alkyl, lower alkyl, and substituted alkylgroups, respectively.

The term “aryl” as used herein, and unless otherwise specified, refersto an aromatic substituent containing a single aromatic ring(monocyclic) or multiple aromatic rings that are fused together, linkedcovalently, or linked to a common group such as a methylene or ethylenemoiety. The common linking group may also be a carbonyl as inbenzophenone, an oxygen atom as in diphenylether, or a nitrogen atom asin diphenylamine Preferred aryl groups contain one aromatic ring or twofused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl,diphenylether, diphenylamine, benzophenone, and the like. “Substitutedaryl” refers to an aryl moiety substituted with one or more substituentgroups, and the terms “heteroatom-containing aryl” and “heteroaryl”refer to aryl in which at least one carbon atom is replaced with aheteroatom. The terms “arylene” and “substituted arylene” refer todivalent aryl and substituted aryl groups as just defined.

The term “heteroatom-containing” as in a “heteroatom-containinghydrocarbyl group” refers to a molecule or molecular fragment in whichone or more carbon atoms is replaced with an atom other than carbon,e.g., nitrogen, oxygen, sulfur, phosphorus or silicon.

“Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 toabout 30 carbon atoms, preferably 1 to about 24 carbon atoms, mostpreferably 1 to about 12 carbon atoms, including branched or unbranched,saturated or unsaturated species, such as alkyl groups, alkenyl groups,aryl groups, and the like. The term “lower hydrocarbyl” intends ahydrocarbyl group of one to six carbon atoms, preferably one to fourcarbon atoms. The term “hydrocarbylene” intends a divalent hydrocarbylmoiety containing 1 to about 30 carbon atoms, preferably 1 to about 24carbon atoms, most preferably 1 to about 12 carbon atoms, includingbranched or unbranched, saturated or unsaturated species, or the like.The term “lower hydrocarbylene” intends a hydrocarbylene group of one tosix carbon atoms, preferably one to four carbon atoms. “Substitutedhydrocarbyl” refers to hydrocarbyl substituted with one or moresubstituent groups, and the terms “heteroatom-containing hydrocarbyl”and “heterohydrocarbyl” refer to hydrocarbyl in which at least onecarbon atom is replaced with a heteroatom. Similarly, “substitutedhydrocarbylene” refers to hydrocarbylene substituted with one or moresubstituent groups, and the terms “heteroatom-containing hydrocarbylene”and “heterohydrocarbylene” refer to hydrocarbylene in which at least onecarbon atom is replaced with a heteroatom. If not otherwise indicated,“hydrocarbyl” indicates unsubstituted hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl, and substitutedheteroatom-containing hydrocarbyl. Unless otherwise indicated, the terms“hydrocarbyl” and “hydrocarbylene” include substituted hydrocarbyl andsubstituted hydrocarbylene, heteroatom-containing hydrocarbyl andheteroatom-containing hydrocarbylene, and substitutedheteroatom-containing hydrocarbyl and substituted heteroatom-containinghydrocarbylene, respectively.

By “substituted” as in “substituted hydrocarbyl,” “substituted alkyl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the hydrocarbyl, alkyl, or other moiety, at least onehydrogen atom bound to a carbon atom is replaced with one or moresubstituents that are functional groups such as alkoxy, hydroxy, halo,nitro, and the like. Unless otherwise indicated, it is to be understoodthat specified molecular segments can be substituted with one or moresubstituents that do not compromise a compound's utility. For example,“succinimidyl” is intended to include unsubstituted succinimidyl as wellas sulfosuccinimidyl and other succinimidyl groups substituted on a ringcarbon atom, e.g., with alkoxy substituents, polyether substituents, orthe like.

The Multifunctional Compound

In accordance with the present invention, a multifunctional compound isprovided that contains a core substituted with a minimum of threereactive groups, each of which participates in a reaction, i.e.,inter-reacts, to form a three-dimensional matrix. The reactive groupsmay be directed attached to the core, or the reactive groups may furthercomprise a linking group through which they are attached to the core.

The reactive groups are selected so that the compound is essentiallynon-reactive in an initial environment. Upon exposure to a specificmodification in the initial environment, providing a modifiedenvironment, the compound is rendered reactive and a plurality ofmultifunctional compounds are then able to inter-react in the modifiedenvironment to form a three-dimensional matrix.

Examples of modification in the initial environment include the additionof an aqueous medium, a change in pH, exposure to ultraviolet radiation,a change in temperature, or contact with a redox initiator. These aredetailed below.

The multifunctional compound is particularly suitable for applicationinvolving contact between a biological system and the multifunctionalcompound and the three-dimensional matrix formed therefrom. Thebiological system can be a biological tissue, and in a preferredembodiment, is living tissue.

The resulting three-dimensional matrix is useful in a variety ofcontexts, and is particularly useful as a biomaterial for medicalapplications, such as for bioadhesion, delivery of biologically activeagents, tissue augmentation, tissue sealing, hemostasis, the preventionof adhesions following a surgical procedure or injury, and so forth.

The core and reactive groups can also be selected so as to provide acompound that has one of more of the following features: arebiocompatible, are non-immunogenic, and do not leave any toxic,inflammatory or immunogenic reaction products at the site ofadministration. Similarly, the core and reactive groups can also beselected so as to provide a resulting matrix that has one or more ofthese features.

In one embodiment of the invention, substantially immediately orimmediately upon exposure to the modified environment, themultifunctional compounds inter-react form a three-dimensional matrix.The term “substantially immediately” is intended to mean within lessthan five minutes, preferably within less than two minutes, and the term“immediately” is intended to mean within less than one minute,preferably within less than 30 seconds.

In one embodiment, the multifunctional compound and resulting matrix arenot subject to enzymatic cleavage by matrix metalloproteinases such ascollagenase, and are therefore not readily degradable in vivo. Further,the multifunctional compound may be readily tailored, in terms of theselection and quantity of each component, to enhance certain properties,e.g., compression strength, swellability, tack, hydrophilicity, opticalclarity, and the like.

The multifunctional compound of the invention is comprised of at leastfour components: a core and a minimum of three reactive groups. In oneembodiment, the multifunctional compound can be described as having theformula (I), where R is the core and the reactive groups are representedby, for example, the reactive group X having an optional linker, L¹:

wherein n is an integer from 1-12, and when n is 2-12, each Z componentmay be different; R is selected from hydrophilic polymers, hydrophobicpolymers, amphiphilic polymers, C₂₋₁₄ hydrocarbyls, andheteroatom-containing C₂₋₁₄ hydrocarbyls; X, Y, and Z are reactivegroups and can be the same or different; L¹, L², and L³ are linkinggroups; and p, q and r are integers from 0-1.

Each of these reactive groups inter-reacts with at least one otherreactive group to form a three-dimensional matrix. Therefore X caninter-react with Y and/or Z, Y can inter-react with X and/or Z, Z caninter-react with X and/or Y and so forth. When n is greater than 1, theindividual Z components may be the same or different, and therefore eachZ group may be the same or different. When the Z groups are different,they can inter-react with each other.

In one preferred embodiment, R is a hydrophilic polymer. In anotherpreferred embodiment, X is a nucleophilic group, Y is an electrophilicgroup, and Z is either an electrophilic or a nucleophilic group.Additional embodiments are detailed below.

A higher degree of inter-reaction, e.g., crosslinking, may be usefulwhen a less swellable matrix is desired or increased compressivestrength is desired. In those embodiments, it may be desirable to have nbe an integer from 2-12. In addition, when a plurality ofmultifunctional compounds are utilized, the compounds may be the same ordifferent.

In the compound of formula (I), each side chain typically has onereactive group. However, the invention also encompasses multifunctionalcompounds where the side chains can contain more than one reactivegroup. Thus, in another embodiment of the invention, the multifunctionalcompound has the formula (II):[X′-(L⁴)_(a)-Y′-(L⁵)_(b)]_(c)-R′  (II)

wherein a and b are integers from 0-1; c is an integer from 3-12; R′ isselected from hydrophilic polymers, hydrophobic polymers, amphiphilicpolymers, C₂₋₁₄ hydrocarbyls, and heteroatom-containing C₂₋₁₄hydrocarbyls; X′ and Y′ are reactive groups and can be the same ordifferent; and L⁴ and L⁵ are linking groups. Each reactive groupinter-reacts with the other reactive group to form a three-dimensionalmatrix. The compound is essentially non-reactive in an initialenvironment but is rendered reactive upon exposure to a modification inthe initial environment that provides a modified environment such that aplurality of the multifunctional compounds inter-react in the modifiedenvironment to form a three-dimensional matrix. In one preferredembodiment, R is a hydrophilic polymer. In another preferred embodiment,X′ is a nucleophilic group and Y′ is an electrophilic group.

The following multifunctional compound shown in the structure of formula(III) is one example of a compound of formula (II):

wherein R⁴ has the formula:

Thus, in formula (III), a and b are 1; c is 4; the core R′ is thehydrophilic polymer, tetrafunctionally activated polyethylene glycol,(C(CH₂—O—)₄; X is the electrophilic reactive group, succinimidyl; Y′ isthe nucleophilic reactive group —CH—NH₂; L⁴ is —C(O)—O—; and L⁵ is—(CH₂—CH₂—O—CH₂)_(x)—CH₂—O—C(O)—(CH₂)₂—.

The multifunctional compounds of the invention are readily synthesizedby techniques that are well known in the art. An exemplary synthesis isset forth below:

A. Reactive Groups

Prior to use, the multifunctional compound is stored in an initialenvironment that insures that the compound remain essentiallynon-reactive until use. Upon modification of this environment, thecompound is rendered reactive and a plurality of compounds will theninter-react to form the desired matrix. The initial environment, as wellas the modified environment, is thus determined by the nature of thereactive groups involved.

The number of reactive groups can be the same or different. However, inone embodiment of the invention, the number of X, Y, and Z reactivegroups are approximately equal. As used in this context, the term“approximately” refers to a 2:1 to 1:2 ratio of moles of one reactivegroup to moles of a different reactive groups. A 1:1:1 molar ratio ofreactive groups is generally preferred.

In general, the concentration of the multifunctional compounds in themodified environment, when liquid in nature, will be in the range ofabout 1 to 50 wt %, generally about 2 to 40 wt %. The preferredconcentration of the compound in the liquid will depend on a number offactors, including the type of compound (i.e., type of molecular coreand reactive groups), its molecular weight, and the end use of theresulting three-dimensional matrix. For example, use of higherconcentrations of the compounds, or using highly functionalizedcompounds, will result in the formation of a more tightly crosslinkednetwork, producing a stiffer, more robust gel As such, compositionsintended for use in tissue augmentation will generally employconcentrations of multifunctional compounds that fall toward the higherend of the preferred concentration range. Compositions intended for useas bioadhesives or in adhesion prevention do not need to be as firm andmay therefore contain lower concentrations of the multifunctionalcompounds.

Electrophilic and Nucleophilic Reactive Groups

In one embodiment of the invention, the reactive groups areelectrophilic and nucleophilic groups, which undergo a nucleophilicsubstitution reaction, a nucleophilic addition reaction, or both. Theterm “electrophilic” refers to a reactive group that is susceptible tonucleophilic attack, i.e., susceptible to reaction with an incomingnucleophilic group. Electrophilic groups herein are positively chargedor electron-deficient, typically electron-deficient. The term“nucleophilic” refers to a reactive group that is electron rich, has anunshared pair of electrons acting as a reactive site, and reacts with apositively charged or electron-deficient site. For such reactive groups,the modification in the initial environment comprises the addition of anaqueous medium and/or a change in pH.

In one embodiment of the invention, X can be a nucleophilic group and Ycan be an electrophilic group or vice versa, and Z can be either anelectrophilic or a nucleophilic group.

X may be virtually any nucleophilic group, so long as reaction can occurwith the electrophilic group Y and also with Z, when Z is electrophilic(Z_(EL)). Analogously, Y may be virtually any electrophilic group, solong as reaction can take place with X and also with Z when Z isnucleophilic (Z_(NU)). The only limitation is a practical one, in thatreaction between X and Y, and X and Z_(EL), or Y and Z_(NU) should befairly rapid and take place automatically upon admixture with an aqueousmedium, without need for heat or potentially toxic or non-biodegradablereaction catalysts or other chemical reagents. It is also preferredalthough not essential that reaction occur without need for ultravioletor other radiation. In one embodiment, the reactions between X and Y,and between either X and Z_(EL) or Y and Z_(NU), are complete in under60 minutes, preferably under 30 minutes. Most preferably, the reactionoccurs in about 5 to 15 minutes or less.

Examples of nucleophilic groups suitable as X or Fn_(NU) include, butare not limited to, —NH₂, —NHR¹, —N(R¹)₂, —SH, —OH, —COOH, —C₆H₄—OH, —H,—PH₂, —PHR', —P(R¹)₂, —NH—NH₂, —CO—NH—NH₂, —C₅H₄N, etc. wherein R¹ is ahydrocarbyl group and each R1 may be the same or different. R¹ istypically alkyl or monocyclic aryl, preferably alkyl, and mostpreferably lower alkyl. Organometallic moieties are also usefulnucleophilic groups for the purposes of the invention, particularlythose that act as carbanion donors. Examples of organometallic moietiesinclude: Grignard functionalities —R²MgHal wherein R² is a carbon atom(substituted or unsubstituted), and Hal is halo, typically bromo, iodoor chloro, preferably bromo; and lithium-containing functionalities,typically alkyllithium groups; sodium-containing functionalities.

It will be appreciated by those of ordinary skill in the art thatcertain nucleophilic groups must be activated with a base so as to becapable of reaction with an electrophilic group. For example, when thereare nucleophilic sulfhydryl and hydroxyl groups in the multifunctionalcompound, the compound must be admixed with an aqueous base in order toremove a proton and provide an —S⁻ or —O⁻ species to enable reactionwith the electrophilic group. Unless it is desirable for the base toparticipate in the reaction, a non-nucleophilic base is preferred. Insome embodiments, the base may be present as a component of a buffersolution. Suitable bases and corresponding crosslinking reactions aredescribed herein.

The selection of electrophilic groups provided on the multifunctionalcompound, must be made so that reaction is possible with the specificnucleophilic groups. Thus, when the X reactive groups are amino groups,the Y and any Z_(EL) groups are selected so as to react with aminogroups. Analogously, when the X reactive groups are sulfhydryl moieties,the corresponding electrophilic groups are sulfhydryl-reactive groups,and the like. In general, examples of electrophilic groups suitable as Yor Z_(EL) include, but are not limited to, —CO—Cl, —(CO)—O—(CO)—R (whereR is an alkyl group), —CH═CH—CH═O and —CH═CH—C(CH₃)═O, halo, —N═C═O,—N═C═S, —SO₂CH═CH₂, —O(CO)—C═CH₂, —O(CO)—C(CH₃)═CH₂, —S—S—(C₅H₄N),—O(CO)—C(CH₂CH₃)═CH₂, —CH═CH—C═NH, —COOH, —(CO)O—N(COCH₂)₂, —CHO,—(CO)O—N(COCH₂)₂—S(O)₂OH, and —N(COCH)₂.

When X is amino (generally although not necessarily primary amino), theelectrophilic groups present on Y and Z_(EL) are amine-reactive groups.Exemplary amine-reactive groups include, by way of example and notlimitation, the following groups, or radicals thereof: (1) carboxylicacid esters, including cyclic esters and “activated” esters; (2) acidchloride groups (—CO—Cl); (3) anhydrides (—(CO)—O—(CO)—R, where R is analkyl group); (4) ketones and aldehydes, including α,β-unsaturatedaldehydes and ketones such as —CH═CH—CH═O and —CH═CH—C(CH₃)═O; (5) halogroups; (6) isocyanate group (—N═C═O); (7) thioisocyanato group(—N═C═S); (8) epoxides; (9) activated hydroxyl groups (e.g., activatedwith conventional activating agents such as carbonyldiimidazole orsulfonyl chloride); and (10) olefins, including conjugated olefins, suchas ethenesulfonyl (—SO₂CH═CH₂) and analogous functional groups,including acrylate (—O(CO)—C═CH₂), methacrylate (—O(CO)—C(CH₃)═CH₂),ethyl acrylate (—O(CO)—C(CH₂CH₃)═CH₂), and ethyleneimino (—CH═CH—C═NH).

In one embodiment the amine-reactive groups contain an electrophilicallyreactive carbonyl group susceptible to nucleophilic attack by a primaryor secondary amine, for example the carboxylic acid esters and aldehydesnoted above, as well as carboxyl groups (—COOH).

Since a carboxylic acid group per se is not susceptible to reaction witha nucleophilic amine, components containing carboxylic acid groups mustbe activated so as to be amine-reactive. Activation may be accomplishedin a variety of ways, but often involves reaction with a suitablehydroxyl-containing compound in the presence of a dehydrating agent suchas dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). Forexample, a carboxylic acid can be reacted with an alkoxy-substitutedN-hydroxy-succinimide or N-hydroxysulfosuccinimide in the presence ofDCC to form reactive electrophilic groups, the N-hydroxysuccinimideester and the N-hydroxysulfosuccinimide ester, respectively. Carboxylicacids may also be activated by reaction with an acyl halide such as anacyl chloride (e.g., acetyl chloride), to provide a reactive anhydridegroup. In a further example, a carboxylic acid may be converted to anacid chloride group using, e.g., thionyl chloride or an acyl chloridecapable of an exchange reaction. Specific reagents and procedures usedto carry out such activation reactions will be known to those ofordinary skill in the art and are described in the pertinent texts andliterature.

Accordingly, in one embodiment, the amine-reactive groups are selectedfrom succinimidyl ester (—O(CO)—N(COCH₂)₂), sulfosuccinimidyl ester(—O(CO)—N(COCH₂)₂—S(O)₂OH), maleimido (—N(COCH)₂), epoxy, isocyanato,thioisocyanato, and ethenesulfonyl.

Analogously, when X is sulfhydryl, the electrophilic groups present on Yand Z_(EL) are groups that react with a sulfhydryl moiety. Such reactivegroups include those that form thioester linkages upon reaction with asulfhydryl group, such as those described in WO 00/62827 to Wallace etal. As explained in detail therein, sulfhydryl reactive groups include,but are not limited to: mixed anhydrides; ester derivatives ofphosphorus; ester derivatives of p-nitrophenol, p-nitrothiophenol andpentafluorophenol; esters of substituted hydroxylamines, includingN-hydroxyphthalimide esters, N-hydroxysuccinimide esters,N-hydroxysulfosuccinimide esters, and N-hydroxyglutarimide esters;esters of 1-hydroxybenzotriazole;3-hydroxy-3,4-dihydro-benzotriazin-4-one;3-hydroxy-3,4-dihydro-quinazoline-4-one; carbonylimidazole derivatives;acid chlorides; ketenes; and isocyanates. With these sulfhydryl reactivegroups, auxiliary reagents can also be used to facilitate bondformation, e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, can beused to facilitate coupling of sulfhydryl groups to carboxyl-containinggroups.

In addition to the sulfhydryl reactive groups that form thioesterlinkages, various other sulfhydryl reactive functionalities can beutilized that form other types of linkages. For example, compounds thatcontain methyl imidate derivatives form imido-thioester linkages withsulfhydryl groups. Alternatively, sulfhydryl reactive groups can beemployed that form disulfide bonds with sulfhydryl groups; such groupsgenerally have the structure —S—S—Ar where Ar is a substituted orunsubstituted nitrogen-containing heteroaromatic moiety or anon-heterocyclic aromatic group substituted with an electron-withdrawingmoiety, such that Ar may be, for example, 4-pyridinyl, o-nitrophenyl,m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2-nitro-4-benzoic acid,2-nitro-4-pyridinyl, etc. In such instances, auxiliary reagents, i.e.,mild oxidizing agents such as hydrogen peroxide, can be used tofacilitate disulfide bond formation.

Yet another class of sulfhydryl reactive groups forms thioether bondswith sulfhydryl groups. Such groups include, inter alia, maleimido,substituted maleimido, haloalkyl, epoxy, imino, and aziridino, as wellas olefins (including conjugated olefins) such as ethenesulfonyl,etheneimino, acrylate, methacrylate, and 4-unsaturated aldehydes andketones.

When X is —OH, the electrophilic functional groups on the remainingcomponent(s) must react with hydroxyl groups. The hydroxyl group may beactivated as described above with respect to carboxylic acid groups, orit may react directly in the presence of base with a sufficientlyreactive electrophilic group such as an epoxide group, an aziridinegroup, an acyl halide, an anhydride, and so forth.

When X is an organometallic nucleophilic group such as a Grignardfunctionality or an alkyllithium group, suitable electrophilicfunctional groups for reaction therewith are those containing carbonylgroups, including, by way of example, ketones, and aldehydes.

It will also be appreciated that certain functional groups can react asnucleophilic or as electrophilic groups, depending on the selectedreaction partner and/or the reaction conditions. For example, acarboxylic acid group can act as a nucleophilic group in the presence ofa fairly strong base, but generally acts as an electrophilic groupallowing nucleophilic attack at the carbonyl carbon and concomitantreplacement of the hydroxyl group with the incoming nucleophilic group.

These, as well as other embodiments are illustrated below, where thecovalent linkages in the matrix that result upon covalent binding ofspecific nucleophilic reactive groups to specific electrophilic reactivegroups on the multifunctional compound include, solely by way ofexample, the following:

TABLE 1 Representative Nucleophilic Group Representative ElectrophilicGroup (X, Z_(NU)) (Y, Z_(EL)) Resulting Linkage —NH₂ —O—(CO)—O—N(COCH₂)₂—NH—(CO)—O— succinimidyl carbonate terminus —SH —O—(CO)—O—N(COCH₂)₂—S—(CO)—O— —OH —O—(CO)—O—N(COCH₂)₂ —O—(CO)— —NH₂ —O(CO)—CH═CH₂—NH—CH₂CH₂—(CO)—O— acrylate terminus —SH —O—(CO)—CH═CH₂—S—CH₂CH₂—(CO)—O— —OH —O—(CO)—CH═CH₂ —O—CH₂CH₂—(CO)—O— —NH₂—O(CO)—(CH₂)₃—CO₂—N(COCH₂)₂ —NH—(CO)—(CH₂)₃—(CO)—O— succinimidylglutarate terminus —SH —O(CO)—(CH₂)₃—CO₂—N(COCH₂)₂—S—(CO)—(CH₂)₃—(CO)—O— —OH —O(CO)—(CH₂)₃—CO₂—N(COCH₂)₂—O—(CO)—(CH₂)₃—(CO)—O— —NH₂ —O—CH₂—CO₂—N(COCH₂)₂ —NH—(CO)—CH₂—O—succinimidyl acetate terminus —SH —O—CH₂—CO₂—N(COCH₂)₂ —S—(CO)—CH₂—O——OH —O—CH₂—CO₂—N(COCH₂)₂ —O—(CO)—CH₂—O— —NH₂—O—NH(CO)—(CH₂)₂—CO₂—N(COCH₂)₂ —NH—(CO)—(CH₂)₂—(CO)—NH—O— succinimidylsuccinamide terminus —SH —O—NH(CO)—(CH₂)₂—CO₂—N(COCH₂)₂—S—(CO)—(CH₂)₂—(CO)—NH—O— —OH —O—NH(CO)—(CH₂)₂—CO₂—N(COCH₂)₂—O—(CO)—(CH₂)₂—(CO)—NH—O— —NH₂ —O—(CH₂)₂—CHO —NH—(CO)—(CH₂)₂—O—propionaldehyde terminus —NH₂

  glycidyl ether terminus —NH—CH₂—CH(OH)—CH₂—O— and—N[CH₂—CH(OH)—CH₂—O—]₂ —NH₂ —O—(CH₂)₂—N═C═O —NH—(CO)—NH—CH₂—O—(isocyanate terminus) —NH₂ —SO₂—CH═CH₂ —NH—CH₂CH₂—SO₂— vinyl sulfoneterminus —SH —SO₂—CH═CH₂ —S—CH₂CH₂—SO₂—

For multifunctional compounds containing electrophilic and nucleophilicreactive groups, the initial environment typically can be dry andsterile. Since electrophilic groups react with water, storage insterile, dry form will prevent hydrolysis. The dry synthetic polymer maybe compression molded into a thin sheet or membrane, which can then besterilized using gamma or e-beam irradiation. The resulting dry membraneor sheet can be cut to the desired size or chopped into smaller sizeparticulates. The modification of a dry initial environment willtypically comprise the addition of an aqueous medium.

In one embodiment, the initial environment can be an aqueous medium suchas in a low pH buffer, i.e., having a pH less than about 6.0, in whichboth electrophilic and nucleophilic groups are non-reactive. Suitableliquid media for storage of such compounds include aqueous buffersolutions such as monobasic sodium phosphate/dibasic sodium phosphate,sodium carbonate/sodium bicarbonate, glutamate or acetate, at aconcentration of 0.5 to 300 mM. Modification of an initial low pHaqueous environment will typically comprise increasing the pH to atleast pH 7.0, more preferably increasing the pH to at least pH 9.5.

In another embodiment the modification of a dry initial environmentcomprises dissolving the multifunctional compound in a first buffersolution having a pH within the range of about 1.0 to 5.5 to form ahomogeneous solution, and (ii) adding a second buffer solution having apH within the range of about 6.0 to 11.0 to the homogeneous solution.The buffer solutions are aqueous and can be any pharmaceuticallyacceptable basic or acid composition. The term “buffer” is used in ageneral sense to refer to an acidic or basic aqueous solution, where thesolution may or may not be functioning to provide a buffering effect(i.e., resistance to change in pH upon addition of acid or base) in thecompositions of the present invention. For example, the multifunctionalcompound can be in the form of a homogeneous dry powder. This powder isthen combined with a buffer solution having a pH within the range ofabout 1.0 to 5.5 to form a homogeneous acidic aqueous solution, and thissolution is then combined with a buffer solution having a pH within therange of about 6.0 to 11.0 to form a reactive solution. For example,0.375 grams of the dry powder can be combined with 0.75 grams of theacid buffer to provide, after mixing, a homogeneous solution, where thissolution is combined with 1.1 grams of the basic buffer to provide areactive mixture that substantially immediately forms athree-dimensional matrix.

Acidic buffer solutions having a pH within the range of about 1.0 to5.5, include by way of illustration and not limitation, solutions of:citric acid, hydrochloric acid, phosphoric acid, sulfuric acid, AMPSO(3-[(1,1-dimethyl-2-hydroxyethyl)amino]2-hydroxy-propane-sulfonic acid),acetic acid, lactic acid, and combinations thereof. In a preferredembodiment, the acidic buffer solution is a solution of citric acid,hydrochloric acid, phosphoric acid, sulfuric acid, and combinationsthereof. Regardless of the precise acidifying agent, the acidic bufferpreferably has a pH such that it retards the reactivity of thenucleophilic groups on the core. For example, a pH of 2.1 is generallysufficient to retard the nucleophilicity of thiol groups. A lower pH istypically preferred when the core contains amine groups as thenucleophilic groups. In general, the acidic buffer is an acidic solutionthat, when contacted with nucleophilic groups, renders thosenucleophilic groups relatively non-nucleophilic.

An exemplary acidic buffer is a solution of hydrochloric acid, having aconcentration of about 6.3 mM and a pH in the range of 2.1 to 2.3. Thisbuffer may be prepared by combining concentrated hydrochloric acid withwater, i.e., by diluting concentrated hydrochloric acid with water.Similarly, this buffer A may also be conveniently prepared by diluting1.23 grams of concentrated hydrochloric acid to a volume of 2 liters, ordiluting 1.84 grams of concentrated hydrochloric acid to a volume to 3liters, or diluting 2.45 grams of concentrated hydrochloric acid to avolume of 4 liters, or diluting 3.07 grams concentrated hydrochloricacid to a volume of 5 liters, or diluting 3.68 grams of concentratedhydrochloric acid to a volume to 6 liters. For safety reasons, theconcentrated acid is preferably added to water.

Basic buffer solutions having a pH within the range of about 6.0 to11.0, include by way of illustration and not limitation, solutions of:glutamate, acetate, carbonate and carbonate salts (e.g., sodiumcarbonate, sodium carbonate monohydrate and sodium bicarbonate), borate,phosphate and phosphate salts (e.g., monobasic sodium phosphatemonohydrate and dibasic sodium phosphate), and combinations thereof. Ina preferred embodiment, the basic buffer solution is a solution ofcarbonate salts, phosphate salts, and combinations thereof.

In general, the basic buffer is an aqueous solution that neutralizes theeffect of the acidic buffer, when it is added to the homogeneoussolution of the compound and first buffer, so that the nucleophilicgroups on the core regain their nucleophilic character (that has beenmasked by the action of the acidic buffer), thus allowing thenucleophilic groups to inter-react with the electrophilic groups on thecore.

An exemplary basic buffer is an aqueous solution of carbonate andphosphate salts. This buffer may be prepared by combining a basesolution with a salt solution. The salt solution may be prepared bycombining 34.7 g of monobasic sodium phosphate monohydrate, 49.3 g ofsodium carbonate monohydrate, and sufficient water to provide a solutionvolume of 2 liter. Similarly, a 6 liter solution may be prepared bycombining 104.0 g of monobasic sodium phosphate monohydrate, 147.94 g ofsodium carbonate monohydrate, and sufficient water to provide 6 liter ofthe salt solution. The basic buffer may be prepared by combining 7.2 gof sodium hydroxide with 180.0 g of water. The basic buffer is typicallyprepared by adding the base solution as needed to the salt solution,ultimately to provide a mixture having the desired pH, e.g., a pH of9.65 to 9.75.

In general, the basic species present in the basic buffer should besufficiently basic to neutralize the acidity provided by the acidicbuffer, but should not be so nucleophilic itself that it will reactsubstantially with the electrophilic groups on the core. For thisreason, relatively “soft” bases such as carbonate and phosphate arepreferred in this embodiment of the invention.

To illustrate the preparation of a three-dimensional matrix of thepresent invention, one may combine an admixture of the multifunctionalcompound with a first, acidic, buffer (e.g., an acid solution, e.g., adilute hydrochloric acid solution) to form a homogeneous solution. Thishomogeneous solution is mixed with a second, basic, buffer (e.g., abasic solution, e.g., an aqueous solution containing phosphate andcarbonate salts) whereupon the reactive groups on the core of themultifunctional compound substantially immediately inter-react with oneanother to form a three-dimensional matrix.

Redox Reactive Groups

In one embodiment of the invention, the reactive groups are vinyl groupssuch as styrene derivatives, which undergo a radical polymerization uponinitiation with a redox initiator. The term “redox” refers to a reactivegroup that is susceptible to oxidation-reduction activation. The term“vinyl” refers to a reactive group that is activated by a redoxinitiator, and forms a radical upon reaction. X, Y, and Z can be thesame or different vinyl groups, for example, methacrylic groups.

For multifunctional compounds containing vinyl reactive groups, theinitial environment typically will be an aqueous environment. Themodification of the initial environment involves the addition of a redoxinitiator.

Oxidative Coupling Reactive Groups

In one embodiment of the invention, the reactive groups undergo anoxidative coupling reaction. For example, X, Y, and Z can be a halogroup such as chloro, with an adjacent electron-withdrawing group on thehalogen-bearing carbon (e.g., on the “L” linking group). Exemplaryelectron-withdrawing groups include nitro, aryl, and so forth.

For such reactive groups, the modification in the initial environmentcomprises a change in pH. For example, in the presence of a base such asKOH, the multifunctional compounds then undergo a de-hydro, chlorocoupling reaction, forming a double bond between the carbon atoms, asillustrated below:

For multifunctional compounds containing oxidative coupling reactivegroups, the initial environment typically can be can be dry and sterile,or a non-basic medium. The modification of the initial environment willtypically comprise the addition of a base.

Photoinitiated Reactive Groups

In one embodiment of the invention, the reactive groups arephotoinitiated groups. For such reactive groups, the modification in theinitial environment comprises exposure to ultraviolet radiation.

In one embodiment of the invention, X can be an azide (—N₃) group and Ycan be an alkyl group such as —CH(CH₃)₂ or vice versa. Exposure toultraviolet radiation will then form a bond between the groups toprovide for the following linkage: —NH—C(CH₃)₂—CH₂—. In anotherembodiment of the invention, X can be a benzophenone(—(C₆H₄)—C(O)—(C₆H₅)) group and Y can be an alkyl group such as—CH(CH₃)₂ or vice versa. Exposure to ultraviolet radiation will thenform a bond between the groups to provide for the following linkage:

For multifunctional compounds containing photoinitiated reactive groups,the initial environment typically will be in an ultravioletradiation-shielded environment. This can be for example, storage withina container that is impermeable to ultraviolet radiation.

The modification of the initial environment will typically compriseexposure to ultraviolet radiation.

Temperature-Sensitive Reactive Groups

In one embodiment of the invention, the reactive groups aretemperature-sensitive groups, which undergo a thermochemical reaction.For such reactive groups, the modification in the initial environmentthus comprises a change in temperature. The term “temperature-sensitive”refers to a reactive group that is chemically inert at one temperatureor temperature range and reactive at a different temperature ortemperature range.

In one embodiment of the invention, X, Y, and Z are the same ordifferent vinyl groups.

For multifunctional compounds containing reactive groups that aretemperature-sensitive, the initial environment typically will be withinthe range of about 10 to 30° C.

The modification of the initial environment will typically comprisechanging the temperature to within the range of about 20 to 40° C.

B. Linking Groups

The reactive groups may be directly attached to the core, or they may beindirectly attached through a linking group, with longer linking groupsalso termed “chain extenders.” In the formula (I) shown above, theoptional linker groups are represented by L¹, L², and L³, wherein thelinking groups are present when p, q and r are equal to 1.

Suitable linking groups are well known in the art. See, for example, WO97/22371 to Rhee et al. Linking groups are useful to avoid sterichindrance problems that can sometimes associated with the formation ofdirect linkages between molecules. Linking groups may additionally beused to link several multifunctional compounds together to make largermolecules. In one embodiment, a linking group can be used to alter thedegradative properties of the compositions after administration andresultant gel formation. For example, linking groups can be used topromote hydrolysis, to discourage hydrolysis, or to provide a site forenzymatic degradation.

Examples of linking groups that provide hydrolyzable sites, include,inter alia: ester linkages; anhydride linkages, such as those obtainedby incorporation of glutarate and succinate; ortho ester linkages; orthocarbonate linkages such as trimethylene carbonate; amide linkages;phosphoester linkages; α-hydroxy acid linkages, such as those obtainedby incorporation of lactic acid and glycolic acid; lactone-basedlinkages, such as those obtained by incorporation of caprolactone,valerolactone, γ-butyrolactone and p-dioxanone; and amide linkages suchas in a dimeric, oligomeric, or poly(amino acid) segment. Examples ofnon-degradable linking groups include succinimide, propionic acid, andcarboxymethylate linkages. See, for example, WO 99/07417 to Coury et al.Examples of enzymatically degradable linkages include Leu-Gly-Pro-Ala,which is degraded by collagenase; and Gly-Pro-Lys, which is degraded byplasmin.

Linking groups can also be included to enhance or suppress thereactivity of the various reactive groups. For example,electron-withdrawing groups within one or two carbons of a sulfhydrylgroup would be expected to diminish its effectiveness in coupling, dueto a lowering of nucleophilicity. Carbon-carbon double bonds andcarbonyl groups will also have such an effect. Conversely,electron-withdrawing groups adjacent to a carbonyl group (e.g., thereactive carbonyl of glutaryl-N-hydroxysuccinimidyl) would increase thereactivity of the carbonyl carbon with respect to an incomingnucleophilic group. By contrast, sterically bulky groups in the vicinityof a reactive group can be used to diminish reactivity and thus reducethe coupling rate as a result of steric hindrance.

By way of example, particular linking groups and corresponding formulasare indicated in Table 2:

TABLE 2 Linking group Component structure —O—(CH₂)_(x)— —O—(CH₂)_(x)-X—O—(CH₂)_(x)-Y —O—(CH₂)_(x)-Z —S—(CH₂)_(x)— —S—(CH₂)_(x)-X—S—(CH₂)_(x)-Y —S—(CH₂)_(x)-Z —NH—(CH₂)_(x)— —NH—(CH₂)_(x)-X—NH—(CH₂)_(x)-Y —NH—(CH₂)_(x)-Z —O—(CO)—NH—(CH₂)_(x)——O—(CO)—NH—(CH₂)_(x)-X —O—(CO)—NH—(CH₂)_(x)-Y —O—(CO)—NH—(CH₂)_(x)-Z—NH—(CO)—O—(CH₂)_(x)— —NH—(CO)—O—(CH₂)_(x)-X —NH—(CO)—O—(CH₂)_(x)-Y—NH—(CO)—O—(CH₂)_(x)-Z —O—(CO)—(CH₂)_(x)— —O—(CO)—(CH₂)_(x)-X—O—(CO)—(CH₂)_(x)-Y —O—(CO)—(CH₂)_(x)-Z —(CO)—O—(CH₂)_(x)——(CO)—O—(CH₂)_(n)-X —(CO)—O—(CH₂)_(n)-Y —(CO)—O—(CH₂)_(n)-Z—O—(CO)—O—(CH₂)_(x)— —O—(CO)—O—(CH₂)_(x)-X —O—(CO)—O—(CH₂)_(x)-Y—O—(CO)—O—(CH₂)_(x)-Z —O—(CO)—CHR²— —O—(CO)—CHR²-X —O—(CO)—CHR²-Y—O—(CO)—CHR²-Z —O—R³—(CO)—NH— —O—R³—(CO)—NH-X —O—R³—(CO)—NH-Y—O—R³—(CO)—NH-Z

In Table 2, x is generally in the range of 1 to about 10; R² isgenerally hydrocarbyl, typically alkyl or aryl, preferably alkyl, andmost preferably lower alkyl; and R³ is hydrocarbylene,heteroatom-containing hydrocarbylene, substituted hydrocarbylene, orsubstituted heteroatom-containing hydrocarbylene) typically alkylene orarylene (again, optionally substituted and/or containing a heteroatom),preferably lower alkylene (e.g., methylene, ethylene, n-propylene,n-butylene, etc.), phenylene, or amidoalkylene (e.g., —(CO)—NH—CH₂).

Other general principles that should be considered with respect tolinking groups are as follows. If a higher molecular weightmultifunctional compound is to be used, it will preferably havebiodegradable linkages as described above, so that fragments larger than20,000 mol. wt. are not generated during resorption in the body. Inaddition, to promote water miscibility and/or solubility, it may bedesired to add sufficient electric charge or hydrophilicity. Hydrophilicgroups can be easily introduced using known chemical synthesis, so longas they do not give rise to unwanted swelling or an undesirable decreasein compressive strength. In particular, polyalkoxy segments may weakengel strength.

C. The Core

The “core” of each multifunctional compound is comprised of themolecular structure to which the reactive groups are bound. Themolecular core can be a polymer, which includes synthetic polymers andnaturally occurring polymers. In one embodiment, the core is a polymercontaining repeating monomer units. The polymers can be hydrophilic,hydrophobic, or amphiphilic. The molecular core can also be a lowmolecular weight component such as a C₂₋₁₄ hydrocarbyl or aheteroatom-containing C₂₋₁₄ hydrocarbyl. The heteroatom-containing C₂₋₁₄hydrocarbyl can have 1 or 2 heteroatoms selected from N, O, and S. In apreferred embodiment, the multifunctional compound comprises a molecularcore of a synthetic hydrophilic polymer.

Hydrophilic Polymers

The term “hydrophilic polymer” as used herein refers to a polymer havingan average molecular weight and composition that naturally renders, oris selected to render the polymer as a whole “hydrophilic.” Preferredpolymers are highly pure or are purified to a highly pure state suchthat the polymer is or is treated to become pharmaceutically pure. Mosthydrophilic polymers can be rendered water-soluble by incorporating asufficient number of oxygen (or less frequently nitrogen) atomsavailable for forming hydrogen bonds in aqueous solutions.

Synthetic hydrophilic polymers may be homopolymers, block copolymersincluding di-block and tri-block copolymers, random copolymers, or graftcopolymers. In addition, the polymer may be linear or branched, and ifbranched, may be minimally to highly branched, dendrimeric,hyperbranched, or a star polymer. The polymer may include biodegradablesegments and blocks, either distributed throughout the polymer'smolecular structure or present as a single block, as in a blockcopolymer. Biodegradable segments preferably degrade so as to breakcovalent bonds. Typically, biodegradable segments are segments that arehydrolyzed in the presence of water and/or enzymatically cleaved insitu. Biodegradable segments may be composed of small molecular segmentssuch as ester linkages, anhydride linkages, ortho ester linkages, orthocarbonate linkages, amide linkages, phosphonate linkages, etc. Largerbiodegradable “blocks” will generally be composed of oligomeric orpolymeric segments incorporated within the hydrophilic polymer.Illustrative oligomeric and polymeric segments that are biodegradableinclude, by way of example, poly(amino acid) segments, poly(orthoester)segments, poly(orthocarbonate) segments, and the like. Otherbiodegradable segments that may form part of the hydrophilic polymercore include polyesters such as polylactide, polyethers such aspolyalkylene oxide, polyamides such as a protein, and polyurethanes. Forexample, the core of the multifunctional compound can be a diblockcopolymer of tetrafunctionally activated polyethylene glycol andpolylactide.

Synthetic hydrophilic polymers that are useful herein include, but arenot limited to: polyalkylene oxides, particularly polyethylene glycol(PEG) and poly(ethylene oxide)-poly(propylene oxide) copolymers,including block and random copolymers; polyols such as glycerol,polyglycerol (PG) and particularly highly branched polyglycerol,propylene glycol; poly(oxyalkylene)-substituted diols, andpoly(oxyalkylene)-substituted polyols such as mono-, di- andtri-polyoxyethylated glycerol, mono- and di-polyoxyethylated propyleneglycol, and mono- and di-polyoxyethylated trimethylene glycol;polyoxyethylated sorbitol, polyoxyethylated glucose; poly(acrylic acids)and analogs and copolymers thereof, such as polyacrylic acid per se,polymethacrylic acid, poly(hydroxyethylmethacrylate),poly(hydroxyethylacrylate), poly(methylalkylsulfoxide methacrylates),poly(methylalkylsulfoxide acrylates) and copolymers of any of theforegoing, and/or with additional acrylate species such as aminoethylacrylate and mono-2-(acryloxy)-ethyl succinate; polymaleic acid;poly(acrylamides) such as polyacrylamide per se, poly(methacrylamide),poly(dimethylacrylamide), poly(N-isopropyl-acrylamide), and copolymersthereof; poly(olefinic alcohols) such as poly(vinyl alcohols) andcopolymers thereof; poly(N-vinyl lactams) such as poly(vinylpyrrolidones), poly(N-vinyl caprolactams), and copolymers thereof;polyoxazolines, including poly(methyloxazoline) andpoly(ethyloxazoline); and polyvinylamines; as well as copolymers of anyof the foregoing. It must be emphasized that the aforementioned list ofpolymers is not exhaustive, and a variety of other synthetic hydrophilicpolymers may be used, as will be appreciated by those skilled in theart.

Those of ordinary skill in the art will appreciate that syntheticpolymers such as polyethylene glycol cannot be prepared practically tohave exact molecular weights, and that the term “molecular weight” asused herein refers to the weight average molecular weight of a number ofmolecules in any given sample, as commonly used in the art. Thus, asample of PEG 2,000 might contain a statistical mixture of polymermolecules ranging in weight from, for example, 1,500 to 2,500 daltonswith one molecule differing slightly from the next over a range.Specification of a range of molecular weights indicates that the averagemolecular weight may be any value between the limits specified, and mayinclude molecules outside those limits Thus, a molecular weight range ofabout 800 to about 20,000 indicates an average molecular weight of atleast about 800, ranging up to about 20 kDa.

Other suitable synthetic hydrophilic polymers include chemicallysynthesized polypeptides, particularly polynucleophilic polypeptidesthat have been synthesized to incorporate amino acids containing primaryamino groups (such as lysine) and/or amino acids containing thiol groups(such as cysteine). Poly(lysine), a synthetically produced polymer ofthe amino acid lysine (145 MW), is particularly preferred. Poly(lysine)shave been prepared having anywhere from 6 to about 4,000 primary aminogroups, corresponding to molecular weights of about 870 to about580,000. Poly(lysine)s for use in the present invention preferably havea molecular weight within the range of about 1,000 to about 300,000,more preferably within the range of about 5,000 to about 100,000, andmost preferably, within the range of about 8,000 to about 15,000.Poly(lysine)s of varying molecular weights are commercially availablefrom Peninsula Laboratories, Inc. (Belmont, Calif.).

Although a variety of different synthetic hydrophilic polymers can beused in the present compounds, preferred synthetic hydrophilic polymersare PEG and PG, particularly highly branched PG. Various forms of PEGare extensively used in the modification of biologically activemolecules because PEG lacks toxicity, antigenicity, and immunogenicity(i.e., is biocompatible), can be formulated so as to have a wide rangeof solubilities, and do not typically interfere with the enzymaticactivities and/or conformations of peptides. A particularly preferredsynthetic hydrophilic polymer for certain applications is a PEG having amolecular weight within the range of about 100 to about 100,000,although for highly branched PEG, far higher molecular weight polymerscan be employed, up to 1,000,000 or more, providing that biodegradablesites are incorporated ensuring that all degradation products will havea molecular weight of less than about 30,000. For most PEGs, however,the preferred molecular weight is about 1,000 to about 20,000, morepreferably within the range of about 7,500 to about 20,000. Mostpreferably, the polyethylene glycol has a molecular weight ofapproximately 10,000.

Naturally occurring hydrophilic polymers include, but are not limitedto: proteins such as collagen, fibronectin, albumins, globulins,fibrinogen, fibrin and thrombin, with collagen particularly preferred;carboxylated polysaccharides such as polymannuronic acid andpolygalacturonic acid; aminated polysaccharides, particularly theglycosaminoglycans, e.g., hyaluronic acid, chitin, chondroitin sulfateA, B, or C, keratin sulfate, keratosulfate and heparin; and activatedpolysaccharides such as dextran and starch derivatives. Collagen andglycosaminoglycans are preferred naturally occurring hydrophilicpolymers for use herein.

The term “collagen” as used herein refers to all forms of collagen,including those, which have been processed or otherwise modified. Thus,collagen from any source may be used in the compounds of the invention;for example, collagen may be extracted and purified from human or othermammalian source, such as bovine or porcine corium and human placenta,or may be recombinantly or otherwise produced. The preparation ofpurified, substantially non-antigenic collagen in solution from bovineskin is well known in the art. For example, U.S. Pat. No. 5,428,022 toPalefsky et al. discloses methods of extracting and purifying collagenfrom the human placenta, and U.S. Pat. No. 5,667,839 to Berg disclosesmethods of producing recombinant human collagen in the milk oftransgenic animals, including transgenic cows. Non-transgenic,recombinant collagen expression in yeast and other cell lines) isdescribed in U.S. Pat. Nos. 6,413,742 to Olsen et al.; 6,428,978 toOlsen et al.; and 6,653,450 to Berg et al.

Collagen of any type, including, but not limited to, types I, II, III,IV, or any combination thereof, may be used in the compounds of theinvention, although type I is generally preferred. Either atelopeptideor telopeptide-containing collagen may be used; however, when collagenfrom a natural source, such as bovine collagen, is used, atelopeptidecollagen is generally preferred, because of its reduced immunogenicitycompared to telopeptide-containing collagen.

Collagen that has not been previously crosslinked by methods such asheat, irradiation, or chemical crosslinking agents is preferred for usein the invention, although previously crosslinked collagen may be used.

Collagens for use in the present invention are generally, although notnecessarily, in aqueous suspension at a concentration between about 20mg/mL to about 120 mg/mL, preferably between about 30 mg/mL to about 90mg/mL. Although intact collagen is preferred, denatured collagen,commonly known as gelatin, can also be used. Gelatin may have the addedbenefit of being degradable faster than collagen.

Nonfibrillar collagen is generally preferred for use in compounds of theinvention, although fibrillar collagens may also be used. The term“nonfibrillar collagen” refers to any modified or unmodified collagenmaterial that is in substantially nonfibrillar form, i.e., molecularcollagen that is not tightly associated with other collagen molecules soas to form fibers. Typically, a solution of nonfibrillar collagen ismore transparent than is a solution of fibrillar collagen. Collagentypes that are nonfibrillar (or microfibrillar) in native form includetypes IV, VI, and VII.

Chemically modified collagens that are in nonfibrillar form at neutralpH include succinylated collagen and methylated collagen, both of whichcan be prepared according to the methods described in U.S. Pat. No.4,164,559 to Miyata et al. Methylated collagen, which contains reactiveamine groups, is a preferred nucleophile-containing component in thecompositions of the present invention. In another aspect, methylatedcollagen is a component that is present in addition to first and secondcomponents in the matrix-forming reaction of the present invention.Methylated collagen is described in, for example, in U.S. Pat. No.5,614,587 to Rhee et al.

Collagens for use in the compositions of the present invention may startout in fibrillar form and can then be rendered nonfibrillar by theaddition of one or more fiber disassembly agent. The fiber disassemblyagent must be present in an amount sufficient to render the collagensubstantially nonfibrillar at pH 7, as described above. Fiberdisassembly agents for use in the present invention include, withoutlimitation, various biocompatible alcohols, amino acids, inorganicsalts, and carbohydrates, with biocompatible alcohols being particularlypreferred. Preferred biocompatible alcohols include glycerol andpropylene glycol. Non-biocompatible alcohols, such as ethanol, methanol,and isopropanol, are not preferred for use in the present invention, dueto their potentially deleterious effects on the body of the patientreceiving them. Preferred amino acids include arginine. Preferredinorganic salts include sodium chloride and potassium chloride. Althoughcarbohydrates, such as various sugars including sucrose, may be used inthe practice of the present invention, they are not as preferred asother types of fiber disassembly agents because they can have cytotoxiceffects in vivo.

Fibrillar collagen is less preferred for use in the compounds of theinvention. However, as disclosed in U.S. Pat. No. 5,614,587 to Rhee etal., fibrillar collagen, or mixtures of nonfibrillar and fibrillarcollagen, may be preferred for use in compounds intended for long-termpersistence in vivo.

Hydrophobic Polymers

The core of the multifunctional compound may also comprise a hydrophobicpolymer, including low molecular weight polyfunctional species; althoughfor most uses hydrophilic polymers are preferred. Generally,“hydrophobic polymers” herein contain a relatively small proportion ofoxygen and/or nitrogen atoms. Preferred hydrophobic polymers for use inthe invention generally have a carbon chain that is no longer than about14 carbons. Polymers having carbon chains substantially longer than 14carbons generally have very poor solubility in aqueous solutions and, assuch, have very long reaction times when mixed with aqueous solutions ofsynthetic polymers containing, for example, multiple nucleophilicgroups. Thus, use of short-chain oligomers can avoid solubility-relatedproblems during reaction. Polylactic acid and polyglycolic acid areexamples of two particularly suitable hydrophobic polymers. Whilecollagen is hydrophobic at neutral pH, it may be made more or lesshydrophilic as through modification with the substituents describedherein.

Amphiphilic Polymers

Generally, amphiphilic polymers have a hydrophilic portion and ahydrophobic (or lipophilic) portion. The hydrophilic portion can be atone end of the core and the hydrophobic portion at the opposite end, orthe hydrophilic and hydrophobic portions may be distributed randomly(random copolymer) or in the form of sequences or grafts (blockcopolymer) to form the amphiphilic polymer core of the multifunctionalcompound. The hydrophilic and hydrophobic portions may include any ofthe aforementioned hydrophilic and hydrophobic polymers.

Alternately, the amphiphilic polymer core can be a hydrophilic polymerthat has been modified with hydrophobic moieties (e.g., alkylated PEG ora hydrophilic polymer modified with one or more fatty chains), or ahydrophobic polymer that has been modified with hydrophilic moieties(e.g., “PEGylated” phospholipids such as polyethylene glycolatedphospholipids).

Low Molecular Weight Components

As indicated above, the molecular core of the multifunctional compoundcan also be a low molecular weight compound, defined herein as being aC₂₋₁₄ hydrocarbyl or a heteroatom-containing C₂₋₁₄ hydrocarbyl, whichcontains 1 to 2 heteroatoms selected from N, O, S, and combinationsthereof. Such a molecular core can be substituted with any of thereactive groups described herein.

Alkanes are suitable C₂₋₁₄ hydrocarbyl molecular cores. Exemplaryalkanes, for substituted with a nucleophilic primary amino group and a Yelectrophilic group, include, ethyleneamine (H₂N—CH₂CH₂—Y),tetramethyleneamine (H₂N₄—CH₄)—Y), pentamethyleneamine (H₂N—(CH₅)—Y),and hexamethyleneamine (H₂N—(CH₆)—Y).

Low molecular weight diols and polyols are also suitable C₂₋₁₄hydrocarbyls and include trimethylolpropane, di(trimethylol propane),pentaerythritol, and diglycerol. Polyacids are also suitable C₂₋₁₄hydrocarbyls, and include trimethylolpropane-based tricarboxylic acid,di(trimethylol propane)-based tetracarboxylic acid, heptanedioic acid,octanedioic acid (suberic acid), and hexadecanedioic acid (thapsicacid).

Low molecular weight di- and poly-electrophiles are suitableheteroatom-containing C₂₋₁₄ hydrocarbyl molecular cores. These include,for example, disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS₃), dithiobis(succinimidylpropionate) (DSP),bis(2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES), and3,3′-dithiobis(sulfosuccinimidylpropionate (DTSPP), and their analogsand derivatives.

In one embodiment of the invention, the multifunctional compound of theinvention comprises a low-molecular weight material core, with aplurality of acrylate moieties and a plurality of thiol groups.

III. Preparation

The multifunctional compounds are readily synthesized to contain ahydrophilic, hydrophobic or amphiphilic polymer core or a low molecularweight core, functionalized with the desired functional groups, i.e.,nucleophilic and electrophilic groups, which enable crosslinking. Forexample, preparation of a multifunctional compound having a polyethyleneglycol (PEG) core is discussed below. However, it is to be understoodthat the following discussion is for purposes of illustration andanalogous techniques may be employed with other polymers.

With respect to PEG, first of all, various functionalized PEGs have beenused effectively in fields such as protein modification (see Abuchowskiet al., Enzymes as Drugs, pp. 367-383 John Wiley & Sons: New York, N.Y.(1981); and Dreborg et al., Crit. Rev. Therap. Drug Carrier Syst. 6:315(1990)), peptide chemistry (see, Mutter et al., The Peptides, Academic:New York, N.Y. 2:285-332; and Zalipsky et al., Int. J. Peptide ProteinRes. 30:740 (1987)), and the synthesis of polymeric drugs (see Zalipskyet al., Eur. Polym. J. 19:1177 (1983); and Ouchi et al., J. Macromol.Sci. Chem. A24:1011 (1987)).

Functionalized forms of PEG, including multi-functionalized PEG, arecommercially available, and are also easily prepared using knownmethods. For example, see Chapter 22 of Poly(ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, J. Milton Harris, ed., PlenumPress, NY (1992).

Multi-functionalized forms of PEG are of particular interest andinclude, PEG succinimidyl glutarate, PEG succinimidyl propionate,succinimidyl butylate, PEG succinimidyl acetate, PEG succinimidylsuccinamide, PEG succinimidyl carbonate, PEG propionaldehyde, PEGglycidyl ether, PEG-isocyanate, and PEG-vinylsulfone. Many such forms ofPEG are described in U.S. Pat. Nos. 5,328,955 and 6,534,591, both toRhee et al. Similarly, various forms of multi-amino PEG are commerciallyavailable from sources such as PEG Shop, a division of SunBio of SouthKorea (www.sunbio.com), Nippon Oil and Fats (Yebisu Garden Place Tower,20-3 Ebisu 4-chome, Shibuya-ku, Tokyo), Nektar Therapeutics (San Carlos,Calif.) and from Huntsman's Performance Chemicals Group (Houston, Tex.)under the name JEFFAMINE® polyoxyalkyleneamines Multi-amino PEGs usefulin the present invention include the JEFFAMINE® diamines (“D” series)and triamines (“T” series), which contain two and three primary aminogroups per molecule. Analogous poly(sulfhydryl) PEGs are also availablefrom Nektar Therapeutics, e.g., in the form of pentaerythritolpoly(ethylene glycol) ether tetra-sulfhydryl (molecular weight 10,000).These multi-functionalized forms of PEG can then be modified to includethe other desired reactive groups.

Reaction with succinimidyl groups to convert terminal hydroxyl groups toreactive esters is one technique for preparing a core with electrophilicgroups. This core can then be modified include nucleophilic groups suchas primary amines, thiols, and hydroxyl groups. Other agents to converthydroxyl groups include carbonyldiimidazole and sulfonyl chloride.However, as discussed herein, a wide variety of electrophilic groups maybe advantageously employed for reaction with corresponding nucleophilicgroups. Examples of such electrophilic groups include acid chloridegroups; anhydrides, ketones, aldehydes, isocyanate, isothiocyanate,epoxides, and olefins, including conjugated olefins such asethenesulfonyl (—SO₂CH═CH₂) and analogous functional groups.

IV. Compositions of the Multifunctional Compound

The multifunctional compound of the invention can be included in apharmaceutical composition. A pharmaceutically acceptable carrier mayalso be included.

In order to enhance matrix strength, it may be generally desirable toadd a “tensile strength enhancer” to the composition. Such tensilestrength enhancers preferably comprise micron-size, preferably 5 to 40microns in diameter and 20 to 5000 microns in length, high tensilestrength fibers, usually with glass transition temperatures well above37° C.

Suitable tensile strength enhancers for use with the multifunctionalcompound of the present invention include, inter alia, collagen fibers,polyglycolide and polylactide fibers, as well as other organic tensilestrength enhancers and inorganic tensile strength enhancers. Aparticularly useful tensile strength enhancer is VICRYL®(polyglycolide:polylactide, 90:10) The use of tensile strengthenhancers, which are part of the broader category of “fillers,” are wellknown. For example, silicone gums, when cross-linked with peroxides, areweak gels with tensile strength on the order of only about 34 N/cm².When suitably compounded with reinforcing fillers, the tensile strengthof these gums may increase as much as fifty-fold. Lichtenwalner et al.,eds., Encyclopedia of Polymer Science and Technology, vol. 12, p. 535(John Wiley, New York, 1970). Suitable tensile strength enhancers arethose that have inherent high tensile strength and also can interact bycovalent or non-covalent bonds with the three-dimensional matrix. Thetensile strength enhancer should bond to the matrix, either mechanicallyor covalently, in order to provide tensile support. Tensile strengths ofpolyglycolide resorbable sutures are approximately 89,000 N/cm²; that ofcollagen fibers is 5000-10,000 N/cm². Tsuruta and Hayashi, eds.,Biomedical Applications of Polymeric Materials (CRC Press, Boca Raton,Fla. 1993).

The multifunctional compound can also be prepared to contain variousimaging agents such as iodine or barium sulfate, or fluorine, in orderto aid visualization of the compositions after administration via X-rayor ¹⁹F-MRI, respectively.

For use in tissue adhesion as discussed below, it may also be desirableto incorporate proteins such as albumin, fibrin, or fibrinogen into themultifunctional compound to promote cellular adhesion.

In addition, the introduction of hydrocolloids such ascarboxymethylcellulose may promote tissue adhesion and/or swellability.

The multifunctional compound may be comprised of a crosslinkablecomposition comprised of (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component comprises two or more amino acid residues selected fromthe group consisting of amino acids comprising primary amine groups andamino acids comprising thiol groups, the second component comprises apolyethylene glycol moiety, and each of the first and secondcrosslinkable components is biocompatible, synthetic, andnonimmunogenic, and further wherein crosslinking of the compositionresults in a biocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, andall m are identical.

In one preferred embodiment, the selected amino acid residues arelysine. Within this embodiment, any of the following is preferred: m>3,m=3, m=4, n=4, the electrophilic groups are succinimidyl moieties, all nare identical, and all m are identical.

In another preferred embodiment, the selected amino acid residues arecysteine. Within this embodiment, any of the following is preferred:m>3, m=3, m=4, n=4, the electrophilic groups are succinimidyl moieties,all n are identical, and all m are identical.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component comprises two or more amino acid residues selected fromthe group consisting of amino acids comprising primary amine groups andamino acids comprising thiol groups, the second component comprises apolyethylene glycol moiety, the electrophilic groups are succinimidylmoieties, and each of the first and second crosslinkable components isbiocompatible, synthetic, and nonimmunogenic, and further whereincrosslinking of the composition results in a biocompatible,nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, andall m are identical.

In one preferred embodiment, the selected amino acid residues arelysine. Within this embodiment, any of the following is preferred: m>3,m=3, m=4, n=4, the electrophilic groups are succinimidyl moieties, all nare identical, and all m are identical.

In another preferred embodiment, the selected amino acid residues arecysteine. Within this embodiment, any of the following is preferred:m>3, m=3, m=4, n=4, the electrophilic groups are succinimidyl moieties,all n are identical, and all m are identical.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component comprises two or more amino acid residues selected fromthe group consisting of amino acids comprising primary amine groups andamino acids comprising thiol groups, the second component comprises amultifunctionally activated polyethylene glycol, and each of the firstand second crosslinkable components is biocompatible, synthetic, andnonimmunogenic, and further wherein crosslinking of the compositionresults in a biocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, allm are identical, the multifunctionally activated polyethylene glycol istetrafunctionally activated polyethylene glycol, and themultifunctionally activated polyethylene glycol is a star-branchedpolyethylene glycol.

In one preferred embodiment, the selected amino acid residues arelysine. Within this embodiment, any of the following is preferred: m>3,m=3, m=4, n=4, the electrophilic groups are succinimidyl moieties, all nare identical, all m are identical, and the multifunctionally activatedpolyethylene glycol is tetrafunctionally activated polyethylene glycolor the multifunctionally activated polyethylene glycol is astar-branched polyethylene glycol.

In another preferred embodiment, the selected amino acid residues arecysteine. Within this embodiment, any of the following is preferred:m>3, m=3, m=4, n=4, the electrophilic groups are succinimidyl moieties,all n are identical, all m are identical, and the multifunctionallyactivated polyethylene glycol is tetrafunctionally activatedpolyethylene glycol or the multifunctionally activated polyethyleneglycol is a star-branched polyethylene glycol.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component comprises two or more amino acid residues selected fromthe group consisting of lysine and cysteine, the second componentcomprises a polyethylene glycol moiety, and each of the first and secondcrosslinkable components is biocompatible, synthetic, andnonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, allm are identical, the first component consists of three lysine residues,and the first component consists of three cysteine residues.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component comprises two or more lysine residues, the secondcomponent comprises a polyethylene glycol moiety, and each of the firstand second crosslinkable components is biocompatible, synthetic, andnonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, allm are identical, and the first component consists of three lysineresidues.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component consists of lysine residues, the second componentcomprises a polyethylene glycol moiety, and each of the first and secondcrosslinkable components is biocompatible, synthetic, andnonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, allm are identical, and the first component consists of three lysineresidues.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5, thefirst component comprises two or more cysteine residues, the secondcomponent comprises a polyethylene glycol moiety, and each of the firstand second crosslinkable components is biocompatible, synthetic, andnonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, allm are identical, and the first component consists of three cysteineresidues.

The multifunctional compound may also be comprised of a crosslinkablecomposition comprised of: (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2; and (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5,wherein the first component consists of cysteine residues, the secondcomponent comprises a polyethylene glycol moiety, and each of the firstand second crosslinkable components is biocompatible, synthetic, andnonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, theelectrophilic groups are succinimidyl moieties, all n are identical, allm are identical, and the first component consists of three cysteineresidues.

The crosslinkable composition may also be comprised of: (a) a firstcrosslinkable component having m nucleophilic groups, wherein m≧2; and(b) a second crosslinkable component having n electrophilic groupscapable of reaction with the m nucleophilic groups to form covalentbonds, wherein n≧2 and m+n≧5, the first component comprises two or moreamino acid residues selected from the group consisting of lysine andcysteine, the second component comprises a polyethylene glycol moiety,the electrophilic groups are succinimidyl moieties, and each of thefirst and second crosslinkable components is biocompatible, synthetic,and nonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, all n areidentical, all m are identical, the first component consists of threelysine residues, and the first component consists of three cysteineresidues.

The crosslinkable composition may also be comprised of: (a) a firstcrosslinkable component having m nucleophilic groups, wherein m≧2; and(b) a second crosslinkable component having n electrophilic groupscapable of reaction with the m nucleophilic groups to form covalentbonds, wherein n≧2 and m+n≧5, the first component comprises two or morelysine residues, the second component comprises a polyethylene glycolmoiety, the electrophilic groups are succinimidyl moieties, and each ofthe first and second crosslinkable components is biocompatible,synthetic, and nonimmunogenic, and crosslinking of the compositionresults in a biocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, all n areidentical, all m are identical, and the first component consists ofthree lysine residues.

The crosslinkable composition may also be comprised of: (a) a firstcrosslinkable component having m nucleophilic groups, wherein m≧2; and(b) a second crosslinkable component having n electrophilic groupscapable of reaction with the m nucleophilic groups to form covalentbonds, wherein n≧2 and m+n≧5, the first component consists of lysineresidues, the second component comprises a polyethylene glycol moiety,the electrophilic groups are succinimidyl moieties, and each of thefirst and second crosslinkable components is biocompatible, synthetic,and nonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, all n areidentical, all m are identical, and the first component consists ofthree lysine residues.

The crosslinkable composition may also be comprised of: (a) a firstcrosslinkable component having m nucleophilic groups, wherein m≧2; and(b) a second crosslinkable component having n electrophilic groupscapable of reaction with the m nucleophilic groups to form covalentbonds, wherein n≧2 and m+n≧5, the first component comprises two or morecysteine residues, the second component comprises a polyethylene glycolmoiety, the electrophilic groups are succinimidyl moieties, and each ofthe first and second crosslinkable components is biocompatible,synthetic, and nonimmunogenic, and crosslinking of the compositionresults in a biocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, all n areidentical, all m are identical, and the first component consists ofthree cysteine residues.

The crosslinkable composition may also be comprised of: (a) a firstcrosslinkable component having m nucleophilic groups, wherein m≧2; (b) asecond crosslinkable component having n electrophilic groups capable ofreaction with the m nucleophilic groups to form covalent bonds, whereinn≧2 and m+n≧5, the first component consists of cysteine residues, thesecond component comprises a polyethylene glycol moiety, theelectrophilic groups are succinimidyl moieties, and each of the firstand second crosslinkable components is biocompatible, synthetic, andnonimmunogenic, and crosslinking of the composition results in abiocompatible, nonimmunogenic, crosslinked matrix.

Any of the following are preferred embodiments of the crosslinkablecomposition described immediately above: m>3, m=3, m=4, n=4, all n areidentical, all m are identical, and the first component consists ofthree cysteine residues.

V. Formation of the Three-Dimensional Matrix

In one embodiment of the invention, a three-dimensional matrix is formedby the steps of: (a) providing a composition comprised of amultifunctional compound of the invention; (b) rendering thenucleophilic and electrophilic groups reactive by exposing thecomposition to an aqueous environment to effect inter-reaction; whereinsaid exposure comprises: (i) dissolving the composition in a firstbuffer solution having a pH within the range of about 1.0 to 5.5 to forma homogeneous solution, and (ii) adding a second buffer solution havinga pH within the range of about 6.0 to 11.0 to the homogeneous solution;and (c) allowing a three-dimensional matrix to form. Typically, thematrix is formed, e.g., by polymerization, without input of any externalenergy.

The first and second components of the composition are typicallycombined in amounts such that the number of nucleophilic groups in themixture is approximately equal to the number of electrophilic groups inthe mixture. As used in this context, the term “approximately” refers toa 2:1 to 1:2 ratio of moles of nucleophilic groups to moles ofelectrophilic groups. A 1:1 molar ratio of nucleophilic to electrophilicgroups is generally preferred.

The first and second components are blended together to form ahomogeneous dry powder. This powder is then combined with a buffersolution having a pH within the range of about 1.0 to 5.5 to form ahomogeneous acidic aqueous solution, and this solution is then combinedwith a buffer solution having a pH within the range of about 6.0 to 11.0to form a reactive solution. For example, 0.375 grams of the dry powdercan be combined with 0.75 grams of the acid buffer to provide, aftermixing, a homogeneous solution, where this solution is combined with 1.1grams of the basic buffer to provide a reactive mixture thatsubstantially immediately forms a three-dimensional matrix.

The buffer solutions are aqueous and can be any pharmaceuticallyacceptable basic or acid composition. The term “buffer” is used in ageneral sense to refer to an acidic or basic aqueous solution, where thesolution may or may not be functioning to provide a buffering effect(i.e., resistance to change in pH upon addition of acid or base) in thecompositions of the present invention.

Acidic buffer solutions having a pH within the range of about 1.0 to5.5, include by way of illustration and not limitation, solutions of:citric acid, hydrochloric acid, phosphoric acid, sulfuric acid, AMPSO(3-[(1,1-dimethyl-2-hydroxyethyl)amino]2-hydroxy-propane-sulfonic acid),acetic acid, lactic acid, and combinations thereof. In a preferredembodiment, the acidic buffer solution is a solution of citric acid,hydrochloric acid, phosphoric acid, sulfuric acid, and combinationsthereof.

Regardless of the precise acidifying agent, the acidic buffer preferablyhas a pH such that it retards the reactivity of the nucleophilic groupson the first component. For example, a pH of 2.1 is generally sufficientto retard the nucleophilicity of thiol groups. A lower pH is typicallypreferred when the first component contains amine groups as thenucleophilic groups. In general, the acidic buffer is an acidic solutionthat, when contacted with nucleophilic groups that are present as partof the first component, renders those nucleophilic groups relativelynon-nucleophilic.

An exemplary acidic buffer is a solution of hydrochloric acid, having aconcentration of about 6.3 mM and a pH in the range of 2.1 to 2.3. Thisbuffer may be prepared by combining concentrated hydrochloric acid withwater, i.e., by diluting concentrated hydrochloric acid with water.Similarly, this buffer A may also be conveniently prepared by diluting1.23 grams of concentrated hydrochloric acid to a volume of 2 liters, ordiluting 1.84 grams of concentrated hydrochloric acid to a volume to 3liters, or diluting 2.45 grams of concentrated hydrochloric acid to avolume of 4 liters, or diluting 3.07 grams concentrated hydrochloricacid to a volume of 5 liters, or diluting 3.68 grams of concentratedhydrochloric acid to a volume to 6 liters. For safety reasons, theconcentrated acid is preferably added to water.

Basic buffer solutions having a pH within the range of about 6.0 to11.0, include by way of illustration and not limitation, solutions of:glutamate, acetate, carbonate and carbonate salts (e.g., sodiumcarbonate, sodium carbonate monohydrate and sodium bicarbonate), borate,phosphate and phosphate salts (e.g., monobasic sodium phosphatemonohydrate and dibasic sodium phosphate), and combinations thereof. Ina preferred embodiment, the basic buffer solution is a solution ofcarbonate salts, phosphate salts, and combinations thereof.

In general, the basic buffer is an aqueous solution that neutralizes theeffect of the acidic buffer, when it is added to the homogeneoussolution of the first and second components and the acid buffer, so thatthe nucleophilic groups of the first component regain their nucleophiliccharacter (that has been masked by the action of the acidic buffer),thus allowing the nucleophilic groups to inter-react with theelectrophilic groups of the second component.

An exemplary basic buffer is an aqueous solution of carbonate andphosphate salts. This buffer may be prepared by combining a basesolution with a salt solution. The salt solution may be prepared bycombining 34.7 g of monobasic sodium phosphate monohydrate, 49.3 g ofsodium carbonate monohydrate, and sufficient water to provide a solutionvolume of 2 liter. Similarly, a 6-liter solution may be prepared bycombining 104.0 g of monobasic sodium phosphate monohydrate, 147.94 g ofsodium carbonate monohydrate, and sufficient water to provide 6 liter ofthe salt solution. The basic buffer may be prepared by combining 7.2 gof sodium hydroxide with 180.0 g of water. The basic buffer is typicallyprepared by adding the base solution as needed to the salt solution,ultimately to provide a mixture having the desired pH, e.g., a pH of9.65 to 9.75.

In general, the basic species present in the basic buffer should besufficiently basic to neutralize the acidity provided by the acidicbuffer, but should not be so nucleophilic itself that it will reactsubstantially with the electrophilic groups of the second component. Forthis reason, relatively “soft” bases such as carbonate and phosphate arepreferred in this embodiment of the invention.

To illustrate the preparation of a three-dimensional matrix of thepresent invention, one may combine an admixture of a first component(e.g., a polyethyleneglycol core with four nucleophilic thiol groups,such as pentaerythritol tetrakis[mercaptoethyl poly(oxyethylene) ether](“HS-PEG”) available from Aldrich Chemical Co. (Milwaukee, Wis.), and asecond component (e.g., a polyethyleneglycol core with fourelectrophilic N-hydroxysuccinimide groups, such as pentaerythritoltetrakis[1-(1′-oxo-5-succimidylpentanoate)-2-poly(oxyethylene) ether](“NHS-PEG,” 10,000 MW, available from Aldrich Chemical Co.), with afirst, acidic, buffer (e.g., an acid solution, e.g., a dilutehydrochloric acid solution) to form a homogeneous solution. Thishomogeneous solution is mixed with a second, basic, buffer (e.g., abasic solution, e.g., an aqueous solution containing phosphate andcarbonate salts) whereupon the first and second components substantiallyimmediately inter-react with one another to form a three-dimensionalmatrix.

VI. Administration

The invention is also directed at a method of formulating an in-situcuring composition. This method involves a delayedactivation/triggering/initiation of the reaction between the reactivegroups, generating a cured composition with consistent and uniformstrength.

The multifunctional compounds of the invention may be administeredbefore, during or after they inter-react in the modified environment toform a three-dimensional matrix. Certain uses, which are discussed ingreater detail below, such as tissue augmentation, may require thematrix to be formed before administration, whereas other applications,such as tissue adhesion, require the compositions to be administeredbefore the inter-reaction has reached “equilibrium.” The point at whichinter-reaction has reached equilibrium is defined herein as the point atwhich the composition no longer feels tacky or sticky to the touch.

The multifunctional compounds of the present invention are generallydelivered to the site of administration in such a way that theindividual reactive groups of the compounds are exposed to the modifiedenvironment for the first time at the site of administration, orimmediately preceding administration. Thus, the compounds are preferablydelivered to the site of administration using an apparatus that allowsthe compounds to be delivered in an initial environment, where thecompounds are essentially non-reactive. For example, a composition canbe delivered to the site so that the individual reactive groups of themultifunctional compound are exposed to an aqueous environment for thefirst time at the site of administration, or immediately precedingadministration. Thus, the composition is delivered using an apparatusthat allows the composition to be delivered in an dry environment, wherethe compounds are essentially non-reactive.

VII. Delivery Systems

A. Multi-Compartment Devices

Suitable delivery systems may involve a multi-compartment spray device,where one or more compartments contain the multifunctional compounds andone or more compartments contain materials needed to provide for themodified environment, so that the multifunctional compounds are exposedto the modified environment as they leave the compartment. Many devicesthat are adapted for delivery of multi-component tissuesealants/hemostatic agents are well known in the art and can also beused in the practice of the present invention. Alternatively, thecompounds can be delivered using any type of controllable extrusionsystem, or they can be delivered manually in the form of pastes,liquids, or dry powders, and exposed to the modified environment at thesite of administration.

The multifunctional compound and the material(s) needed to provide forthe modified environment may be conveniently formed under asepticconditions by placing each of the ingredients into separate syringebarrels. For example, the multifunctional compound and the material(s)needed to provide for the modified environment can be housed separatelyin a multiple-compartment syringe system having a multiple barrels, amixing head, and an exit orifice. Material(s) can then be added to thebarrel housing the multifunctional compound, which is then extruded intothe mixing head. Additional materials can be simultaneously extrudedinto the mixing head, if needed. Finally, the resulting composition canthen be extruded through the orifice onto a surface.

For example, the syringe barrels holding the multifunctional compoundand other material(s) may be part of a dual-syringe system, e.g., adouble barrel syringe as described in U.S. Pat. No. 4,359,049 to Redl etal. In this embodiment, the acid buffer can be added to the syringebarrel that also holds the multifunctional compound in e.g., a drypowder, so as to produce the homogeneous solution. In other words, theacid buffer may be added (e.g., injected) into the syringe barrelholding the dry powder to thereby produce a homogeneous solution of thefirst and second components. This homogeneous solution can then beextruded into a mixing head, while the basic buffer is simultaneouslyextruded into the mixing head. Within the mixing head, the homogeneoussolution and the basic buffer are mixed together to thereby form areactive mixture. Thereafter, the reactive mixture is extruded throughan orifice and onto a surface (e.g., tissue), where a film is formed,which can function as a sealant or a barrier, or the like. The reactivemixture begins forming a three-dimensional matrix immediately upon beingformed by the mixing of the homogeneous solution and the basic buffer inthe mixing head. Accordingly, the reactive mixture is preferablyextruded from the mixing head onto the tissue very quickly after it isformed so that the three-dimensional matrix forms on, and is able toadhere to, the tissue.

A preferred embodiment of the multi-compartment syringe system of thepresent invention is shown in FIG. 1. The device is comprised of threesyringes, two housing each of the two buffers of the present inventionwith the third syringe housing the multifunctional compound in drypowder form 1. The two syringes housing the solutions 1 arepre-assembled into a syringe housing 2 with a transfer port closure 3attached to the housing assembly 2 to allow mixing of themultifunctional compound into the correct syringe. A syringe clip 4 isattached to the plunger rod of the syringe that does not require mixingwith the dry powder multifunctional compound.

Other systems for combining reactive materials are well known in theart, and include the systems described in U.S. Pat. Nos. 6,454,786 toHolm et al.; 6,461,325 to Delmotte et al.; 5,585,007 to Antanavich etal.; 5,116,315 to Capozzi et al.; 4,631,055 to Redl et al.; and U.S.Patent Application Publication No. 2004/0068266 to Delmotte.

B. Pressurized Delivery Devices

Other delivery systems for dispensing the multicomponent compositions ofthe invention may include pressurized delivery devices, examples ofwhich are described in commonly owned co-pending U.S. patent applicationSer. No. 10/957,493, filed on Oct. 1, 2004, andentitled “Mixing andDispensing Fluid Components of a Multicomponent Composition.” Such apressurized delivery device may include a diffuser surface having anoutlet extending therethrough that is positioned downstream from aplurality of inlets. While at least one inlet is adapted to communicatewith a source of a pressurized carrier fluid, each of a plurality ofinlets is adapted to communicate with a source of a different fluidcomponent. Using this device, the multifunctional compound in a drypowder form is premixed with the first buffer to form a homogeneoussolution as previously described and this solution is subsequentlycommunicated with a first fluid component. The second fluid componentwill communicate with the second buffer solution previously described.Once the diffuser surface receives fluid components from the inlets,each received fluid component is pushed toward the outlet for mixing anddispensing therethrough by the pressurized carrier fluid, typically agas such as air, from the carrier fluid inlet. The diffuser surface andthe inlets may represent components of a mixing nozzle.

In general, there are two categories of gas-enhanced nozzles fordispensing reactive components of a multicomponent composition—thosethat involve internal mixing and those that involve external mixing.When the diffuser surface is a part of a nozzle, the nozzle may beconsidered an internal-mixing nozzle. Unlike other internal-mixingtechnologies, the internal-mixing nozzle of the pressurized deliverydevice of the present invention provides several features that serveindividually and collectively to eliminate clogging. For example, adiffuser surface typically has a shape effective to direct and maintaineach received fluid component in a different flow path on the diffusersurface toward the outlet for mixing therein and dispensingtherethrough. Due to the minimal residence time of the mixture withinthe nozzle, reactive components do not have time to set and clog thenozzle before the mixture is forced out of the nozzle by the pressurizedcarrier fluid. In addition, the outlet may be aligned with any or all ofthe carrier fluid inlets that may be present in the nozzle to direct thepressurized carrier fluid in a manner that enhances fluid componentmixing and to expel the mixture in a jet like manner. As the orientationof the diffuser surface relative to the inlets affects the performanceof the device, the diffuser surface may be permanently affixed orimmobilized with respect to the inlets; however, when the diffusersurface is detachable from the inlets, the nozzle may be disassembled tofacilitate cleaning and/or replacement of parts. For example, thediffuser surface may be replaceable/and or disposable. Nevertheless,when the pressurized delivery device of the present invention hasdiffuser surface that is detachable from the inlets, the device may beconstructed to allow assembly of the components in only configurationsthat align the diffuser surface to the inlets such that the performanceof the device is optimized.

FIGS. 2 and 3 illustrate an example of the pressurized delivery deviceof the present invention in the form of a nozzle that includes all ofthe above-discussed features which serve eliminate the problemsassociated with nozzle clogging. As is the case with all figuresreferenced herein, in which like parts are referenced by like numerals,FIGS. 2 and 3 are not necessarily to scale, and certain dimensions maybe exaggerated for clarity of presentation. As depicted in FIG. 2, thenozzle 1 includes a cap 10 having a slot-shaped outlet orifice 12 thatextends through the center of the distal end 14 of the cap 10. The cap10 is shown as having a cylindrical exterior surface 16 and an interiorsurface 18 that terminates at opening 20, but additional cap shapes arealso suitable for use with the pressurized delivery device of thepresent invention. As shown in FIG. 3, the interior surface 18 of thecap 10 at end 14 serves to receive fluid components thereon.

Also provided is a generally elongate cylindrical connector 30 in theform of a unitary member having a first terminus 32 and a secondterminus 34. A plurality of inlet lumens 36A, 36B, and 36C traversingthe length of the connector defined by termini 32 and 34. As depicted,the connector 30 is detached from the cap 10. Inlet lumens 36A and 36Beach communicate at the second terminus 34 with a different source of afluid component, e.g., the first buffer mixed with the dry powder in onesource and the second buffer in the other source (not shown). Similarly,inlet lumens 36C are provided fluid communication at the second terminus34 with a source of pressurized carrier gas (not shown). The carrierfluid inlet lumens 36C define a plane that is perpendicular to a planedefined by the fluid component inlet lumens 36A and 36B. As depicted inFIGS. 2 and 3, the first terminus 32 of the connector 30 has dimensionssuitable for forming a fluid-tight seal against the interior surface 18of the cap 10 at its proximal end 20.

In operation, the cap 10 is placed over the first terminus 32 of theconnector 30 such that the carrier fluid inlet lumens 36C are alignedwith outlet orifice 12. In addition, each of a plurality of differentfluid component sources is provided fluid communication with the fluidcomponent inlet lumens 36A and 36B and at least one source ofpressurized carrier gas is provided fluid communication with the carrierfluid inlet 36C.

As discussed above, the interior surface 18 of the cap 10 at distal end14 serves as a diffuser surface 18 that is adapted to receive fluidcomponents thereon. As depicted in FIG. 2, the diffuser surface 18exhibits two-fold axial symmetry. The dotted lines indicate the positionof lumens 36A, 36B, and 36C relative to the diffuser surface 18.Similarly, in FIG. 2, the dashed lines shown within the connector 30indicate the separate flow paths of the fluid components emerging fromthe fluid component inlet lumens 36A and 36B, respectively, and directedby the diffuser surface 18 in a generally inward direction toward thecenter outlet orifice 12. Once the fluid components reach outlet orifice12, pressurized gas from carrier fluid lumens 36C mix the fluidcomponents and force the mixture out of the outlet orifice 12.

In the pressurized delivery device of the present invention, thegeometries of, and spatial relationships between the various componentsof diffuser surface 18 represent an important aspect of the pressurizeddelivery device. For example, the pressurized delivery device may beused to carry out mixing of a plurality of reactive components.Typically, nozzles for mixing reactive components are of the externalmixing category because previously known internal mixing designs areprone to clogging. Clogging results when reactive components are mixedprior to introduction to the gas stream. In contrast, the pressurizeddelivery device provides a high-pressure area between the inlets 36A-36Cand the diffuser surface 18 that serves to mix reactive fluids whilesimultaneously forcing the mixture out of the orifice 12.

In addition, the diffuser surface 18 is located downstream from theinlets 36A-36C and is effective to direct fluid components toward theoutlet for mixing and dispensing therethrough by a pressurized carrierfluid; thus, the diffuser surface 18 should exhibit an appropriate shapeto carry out its desired function while minimizing the odds of deviceclogging. For example, while the diffuser surface 18 depicted in FIGS. 2and 3 is located within a cylindrical cap 10 having a flat exteriorcircular end surface and contains a centrally located slot-shapedorifice 12, such geometry is not required.

As depicted in FIGS. 2 and 3, the exterior surface of the cap isparallel to the diffuser surface. While such a parallel configuration ofthe exterior surface of the cap is preferred, it is understood that itis merely exemplary and not a requirement of the pressurized deliverysystem of the present invention. Similarly, while both FIGS. 2 and 3depict caps that exhibit axial symmetry, such axial symmetry is merelypreferred and not required. Where the caps of the present invention aresymmetrical, the symmetry may be axial or mirror symmetry. It isexpected that variations on diffuser surface shapes and nozzleconfigurations may be developed through routine experimentation. Withrespect to the inlets, the pressurized delivery device of the presentinvention generally requires a plurality of fluid component inlets forcommunication with an equal or less number of sources of fluidcomponents. While a single carrier fluid inlet may be provided, thepressurized delivery device typically provides a plurality of carrierfluid inlets. Often the carrier fluid inlets are provided communicationwith a single source of carrier fluid via a splitter or manifold, thougha plurality of carrier fluid sources may be advantageously used as wellin certain instances.

In addition, inlets are typically each located at the terminus of acorresponding lumen. In some instances, the lumens may coextend throughan elongate cylindrical connector 30, as depicted in FIG. 2 (with36A-36C depicting the lumens). Alternatively, the lumens may extendthrough separate tubes. Furthermore, tubes and/or tubing members may beconstructed to form a lumen assembly. For example, various lengths ofmultilumen delivery tubing for use in specific surgical or non-surgicalapplications. Particularly in laparoscopic applications, it may beuseful to employ flexible tubing. The tubing serves to maintainseparation of the two fluid components and provide a pathway for thedelivery of pressurized gas to the diffuser surface.

Additional features also serve to enhance the mixing and deliveryperformance of the pressurized delivery device of the present invention.As discussed above, two or more fluid components may be individuallydelivered through inlets to impinge upon the diffuser surface.Typically, the components first impinge upon the diffuser plate near theoutlet to reduce the residence time of the components in the device. Anyof a number of means may be used to provide motive force to introducefluid components through the inlets and toward the outlet. Exemplarymotive force means include pumps, compressors, pistons, and the like.

Then, as the diffuser plate directs the components toward the outlet 12,and the pressurized carrier fluid simultaneously provides a force to mixand expel the components through the outlet. Accordingly, one or morethe carrier fluid inlets are positioned such that a high-pressure zoneis created between the component inlets and the diffuser surface while acomparatively low-pressure zone is created downstream from the outlet.“Dead space” that serve to trap residue is generally avoided. As aresult, a fluid mixture is forced through the outlet in a jet-likefashion, thereby reducing any potential or actual buildup of residuethat serve to clog the pressurized delivery device.

In general, any of a number of carrier fluids may be employed with thepressurized delivery device of the present invention. For example, thecarrier fluid may be gaseous and/or liquid in nature. Typically,however, the carrier fluid is chemically inert with respect to the fluidcomponents. Suitable inert gases include, without limitation, air,carbon dioxide, nitrogen, argon, helium, gaseous perfluorinated alkanesand ethers, gaseous chlorofluorocarbons and the like. Suitable inertliquids include, without limitation, polysiloxanes, perfluoroinatedpolyethers, and the like. Pressurized air represents an economical andpractical carrier fluid for use with the pressurized delivery device.Equipment associated with pressurized air is well known in the art andmay include pressurized tanks or cylinders as well as compressors. Insome instances, one or more check valves, e.g., one-way valves, may beprovided to prevent back flow of fluid component resulting from pressurebuildup associated with the use of the pressurized delivery device. Suchcheck valves may be positioned upstream from the diffuser surface, e.g.,within lumens associated with the inlets. Such check valves areparticularly useful when inlet lumens are short, e.g., about 2 to about5 centimeters in length, because the potential for back flow tends to beinversely proportional to the length of the lumens; however, checkvalves may be employed with longer lumens as well.

The portions of the device that contact multicomponent composition andthe fluid components thereof should be inert and preferably repellant tothe materials contacted. Thus, portions of the device that contact thefluids in operation should be selected according to the fluidsthemselves. For example, the device or components thereof may be madefrom a plastic such as polycarbonates, polyurethane, polyesters,acrylics, ABS polymers, polysulfone, and the like. Adhesion inhibitingcoatings such as polysiloxanes, perfluorinated polymers, and the likemay be used as well. Thus, the diffuser surface is typically inert andoptionally repelling to the fluid components. Similar, lumen surfacesthat may contact the fluid components or the carrier fluid are typicallyinert and optionally repelling to the corresponding fluid as well.

The pressurized delivery device of the present invention is particularlyuseful for dispensing multicomponent compositions. While some gaseouscomponents may be used, the pressurized delivery device is particularlyuseful for liquids. Thus, at least one fluid component is usually aliquid. Often, each fluid component includes a liquid. For example, thepressurized delivery device is useful to dispense composition such asfluid mixtures, wherein the mixing of a plurality of fluids results inan increase in viscosity sufficient to impair mixture flow. Suchcompositions may be formed from fluid components that are chemicallyreactive with respect to each other. In some instances, a crosslinkingagent may be provided.

In practice, then, a diffuser surface having an outlet extendingtherethrough such that the diffuser surface is downstream from aplurality of fluid component inlets and at least one carrier fluidinlet. A different fluid component is directed from each of the fluidcomponent inlets toward the diffuser surface. In some instances, fluidcomponents are directed at substantially the same flow rate toward thediffuser surface. Alternatively, the fluid components are directed atdifferent flow rates toward the diffuser surface. Typically, the flowrate of the carrier fluid is higher than that for the fluid components.The diffuser surface maintains and directs each received fluid componentin a different flow path toward the outlet. Pressurized carrier fluidfrom the at least one carrier fluid inlet is also directed through theoutlet, thereby mixing the fluid components present at the outlet anddispensing the composition through the outlet.

C. Other Delivery Systems

Yet another way of delivering the multifunctional compounds of thepresent invention is to prepare the compounds in such a manner so thatthe individual reactive groups of the compounds are in an inactive formas either a liquid or powder. Such reactive groups can then be activatedafter application to the tissue site, or immediately beforehand, byapplying an activator, which provides for the modified environment. Forexample, the activator can be a buffer solution having a pH that willactivate the reactive groups on the compounds once mixed therewith.Still another way of delivering the compositions is to prepare preformedsheets, and apply the sheets as such to the site of administration.

VIII. Kits

The multifunctional compounds of the invention can also be packaged inkits and used in a variety of medical applications. The kit wouldinclude a plurality of multifunctional compounds, as well as whatevermaterials are needed to change the environment so as to render thecompound reactive, e.g., buffer solutions, as well as written orotherwise illustrated instructions for use. For example a typical kitfor use in medical applications, may include: (a) a multifunctionalcompound of the invention or a plurality of multifunctional compounds ofthe invention; (b) a first buffer solution having a pH within the rangeof about 1.0 to 5.5; and (c) a second buffer solution having a pH withinthe range of about 6.0 to 11.0; wherein each component is packagedseparately and admixed immediately prior to use. As is evident to thoseof ordinary skill in the art, prior to use, each component should remainin a separate sterile package. As previously described where thereactive groups of the multifunctional compound are nucleophilic andelectrophilic groups, the nucleophilic and electrophilic groups arenon-reactive in a dry environment, but are rendered reactive uponexposure to an aqueous environment such that a plurality of themultifunctiona compounds interact in the aqueous environment to form athree-dimensional matrix.

In another embodiment, the kit can further comprise a delivery systemthat will allow the composition to be delivered as a spray. The spraycan be generated by manually mixing the components and passing themthrough a spray nozzle. The spray generation can also be accomplished byusing a flow of gas (for example, air, nitrogen, carbon dioxide).

Kits contemplated under the present invention will preferably include adelivery system for the compositions of the present invention. Deliverydevices that may be included in the kits will preferably be one of themulti-component syringe device and/or the pressurized delivery devicesdescribed herein.

In one embodiment of the kit, a multi-component syringe device isincluded in the kit. As previously described, the multi-component spraydevice may be a multiple-compartment syringe system having multiplebarrels, a mixing head, and an exit orifice, wherein the dry powdercomposition, the first buffer, and the second buffer are housedseparately in the multiple-compartment syringe system.

FIG. 1 describes a preferred embodiment of the multi-compartment device.When provided in a kit, the device is provided with three pouches. Thefirst pouch is a liquid components pouch, which consists of two syringesthat are preassembled into a housing. A transfer port closure isattached to the housing assembly to allow mixing of the dry powders intothe correct syringe. A clip is attached to the plunger rod of thesyringe that does not require mixing with the dry powders. The secondpouch is a powder component pouch, which consists of a syringecontaining the dry powder(s) and a dessicant package. The third pouch isan applicator pouch, which contains two applicators.

To use the preferred kit of FIG. 1, each pouch is opened using aseptictechniques and the contents of each pouch are transferred into a sterilefield. In the sterile field, the liquid and powder components areprepared as follows. Without removing the syringe clip, the luer cap onthe transfer port closure is removed. The cap is removed from the powdersyringe and the powder syringe is connected to the opening of thetransfer port closure. The liquid is transferred into the powder byforcefully depressing the plunger. The contents between the two syringesare mixed back and forth between the two syringes until the solid iscompletely dissolved (e.g., 18-20 times). The entire content is thenpushed into the syrnting contained in the syringe housing. The powdersyringe is disengaged by detaching the transfer port closure by graspingthe powder syringe barrel; pressing the levers on the syringe housing;and pulling both the empty powder syringe and transfer port closure fromthe housing. To expel all air from the syringe, the syringe tips areheld up, the syringe plungers are leveled, the syringe clip is rotatedto connect to the other plunger; and holding the syringe upright, allair is expelled from the syringe. As a final step, the applicator issnapped onto the end of the syringe housing making the composition readyto use. A clear gel should be seen approximately three minutes followingthe mixing of the components.

In another embodiment of the kit, a pressurized delivery device isincluded in the kit. As previously described, the pressurized deliverydevice of the present invention includes a plurality of fluid componentinlets each adapted to communicate with a source of different fluidcomponents; at least one carrier fluid inlet adapted to communicate witha source of a pressurized carrier fluid; a diffuser surface locateddownstream from the plurality of fluid component inlets and the at leastone carrier fluid inlet; and an outlet extending through the diffusersurface, wherein the diffuser surface is adapted to receive fluidcomponents thereon and has a shape effective to direct and maintain eachreceived fluid component in a different flow path toward the outlet formixing and dispensing therethrough by the pressurized carrier fluid fromthe at least one carrier fluid inlet.

Kits contemplated under the present invention are not limited to thedevices described herein and may also include any other suitabledelivery device known in the art of drug delivery.

Exemplary medical applications involve, by way of illustration and notlimitation, adhering or sealing biological tissue, delivering abiologically active agent (in which case, the kit would further comprisea biologically active agent, for example, mixed with the components toform a homogeneous mixture or packaged separately), delivering cells andgenes (in which case, the kit would further comprise the living cells orgenes, for example, mixed with the components to form a homogeneousmixture or packaged separately), bioadhesion, in ophthalmicapplications, for tissue augmentation, for adhesion prevention, formingsynthetic implants and coating synthetic implants, and for the treatmentof aneurysms. In a preferred embodiment, the mixture of the biologicallyactive agents with the components is a homogeneous mixture; however,this feature is not required. Whether packaged together or separately,each of the components and the biological agent should be in sterilepackages prior to use.

For purposes of description only, the surgical use of themulti-compartment syringe kit of FIG. 1 is described. As a preliminarystep, blood circulation to the surgical site is restored to expand thegraft by unclamping the site and after circulation is restored, the siteis reclamped to stop circulation. Excess blood is aspirated and allsurfaces are air dried prior to application of the composition. Holdingthe applicator approximately 3 cm from the site (touching the site orholding more than 6 cm from the site is not recommended), sealant isforcibly applied to the site. To enhance mixing, the applicator is movedquickly along the anastomotic site. If the composition is to be appliedto more than one site, the applicator tip should be wiped with gauze andthe device should be set upright to prevent clogging. If the compositiondoes not gel within 30 seconds, i.e., the composition remains watery onthe site, the site should be flushed with saline and the materialaspirated. If the treated site fails to seal, the surface should beblotted dry; reclamping the vessel may be required to dry the field forreapplication of the composition. If the applicator becomes clogged, itshould be replaced with a new applicator.

IX. Uses

The multifunctional compounds of the present invention can be used in avariety of different applications. In general, the compounds can beadapted for use in any tissue engineering application where syntheticgel matrices are currently being utilized. For example, the compositionsare useful as tissue sealants, vascular sealants, in tissueaugmentation, in tissue repair, as hemostatic agents, in preventingtissue adhesions, in providing surface modifications, and indrug/cell/gene delivery applications and may be used in a variety ofopen, endocopic, and laparoscopic surgical procedures. One of skill inthe art can easily determine the appropriate administration protocol touse with any particular composition having a known gel strength andgelation time. A more detailed description of several specificapplications is given below.

A. Tissue Sealants and Adhesives

In one application, the multifunctional compounds described herein canbe used for medical conditions that require a coating or sealing layerto prevent the leakage of gases, liquid, or solids. The method entailsapplying the multifunctional compounds to the damaged tissue or organ toseal 1) vascular and or other tissues or organs to stop or minimize theflow of blood; 2) thoracic tissue to stop or minimize the leakage ofair; 3) gastrointestinal tract or pancreatic tissue to stop or minimizethe leakage of fecal or tissue contents; 4) bladder or ureters to stopor minimize the leakage of urine; 5) dura to stop or minimize theleakage of CSF; and 6) skin or serosal tissue to stop the leakage ofserosal fluid. These compounds may also be used to adhere biologicaltissues together such as small vessels, nerves, or dermal tissue. Suchbiological tissues will typically, but not necessarily, be livingtissue. The compounds can be used 1) by applying them to the surface ofone tissue and then a second tissue may be rapidly pressed against thefirst tissue or 2) by bringing the tissues in close juxtaposition andthen applying the compounds. In addition, the compounds can be used tofill spaces in soft and hard tissues that are created by disease orsurgery.

In one embodiment of the invention, there is provided a method ofsealing tissue of a patient comprising the steps of: (a) providing acomposition comprising a plurality of the multifunctional compounds inan initial environment; (b) rendering the reactive groups reactive byexposing the compound to a modified environment; and (c) placing the aplurality of the multifunctional compounds into contact with tissue andallowing a three-dimensional matrix to form and seal the tissue.

In another embodiment of the invention, there is provided a method ofsealing tissue of a patient comprising the steps of: (a) providing acomposition of the invention comprising a plurality of themultifunctional compounds of the invention; (b) rendering thenucleophilic and electrophilic groups reactive by exposing thecomposition to an aqueous environment to effect inter-reaction; whereinsaid exposure comprises: (i) dissolving the composition in a firstbuffer solution having a pH within the range of about 1.0 to 5.5 to forma homogeneous solution, and (ii) adding a second buffer solution havinga pH within the range of about 6.0 to 11.0 to the homogeneous solutionto form a mixture; and (c) placing the mixture into contact with tissueand allowing a three-dimensional matrix to form and seal the tissue.

In a further embodiment of the invention, the compositions can beapplied in conjunction with an implanted medical device such that itprevents the leakage of gases, liquids or solids from the device or fromthe device-tissue interface. For example, following the implantation ofa vascular graft (either synthetic or biological), there is oftenleakage of blood through the suture holes in the graft or at theinterface between the graft and the tissue. The composition of theinvention can be applied to this area to prevent further blood leakage.

In certain aspects of the invention, the composition may be furthercombined with a fibrosing agent to further enhance the properties of thesealant or adhesive. In one aspect of the present invention, a fibrosing(i.e., scarring) agent can be included in a polymeric sealant spraywhich solidifies into a film or coating to promote fibrosis and seal airleaks.

In one illustrative application, a fibrosing agent may be included withthe polymer composition for use as a pulmonary sealant during open orendoscopic lung reduction surgeries, for example, to seal off pulmonarybullae in open and endoscopic lung destruction procedures. The additionof a fibrosis-inducing agent to a pulmonary sealant can induce theformation of a stable, fibrous scar that permanently seals the parietalsurface of the lung at the surgical location (or the alveolar surface ofthe lung if delivered endoscopically during lung reduction surgery),reduces hospitalization time, and prevents recurrence of the air leak.Clinically a fibrosis-inducing pulmonary sealant can be useful toimprove the outcomes in open lung surgery, endoscopic lung reductionsurgery for emphysema (severe COPD), esophageal leaks after endoscopy orresection, complications of treatment of other intra-thoracicmalignancies, pleural effusion, haemothorax, pneumothorax, chylothorax,complications of aspiration, and tuberculosis.

It should be apparent to one of skill in the art that potentially anyadhesion or fibrosis-inducing agents described above may be utilizedalone, or in combination with the present composition, in the practiceof this embodiment. Exemplary fibrosing agents for use in sealants andadhesive include talc, silk, wool, chitosan, polylysine, fibronectin,bleomycin, and connective tissue growth factor (CTGF), as well asanalogues and derivatives of the aforementioned.

The exact dose administered can vary with the composition of the sealantor adhesive; however, certain principles can be applied in theapplication of this art. Drug dose can be calculated as a function ofdose per unit area (of the amount of the sealant being applied), totaldrug dose administered can be measured, and appropriate surfaceconcentrations of active drug can be determined Regardless of the methodof incorporation of the drug into the sealant or adhesive, the exemplaryfibrosing agents, used alone or in combination, should be administeredunder the following dosing guidelines:

Utilizing talc as an exemplary fibrosis-inducing agent, the total doseof talc delivered from a sealant, or coated onto the surface of a lung,should not exceed 100 mg (range of 1 μg to 100 mg). In one embodiment,the total amount of talc released from the sealant should be in therange of 10 μg to 50 mg. The dose per unit area (i.e., the dosage oftalc as a function of the surface area of the lung to which drug isapplied) should fall within the range of 0.05 μg-10 μg per mm² ofsurface area coated. In another embodiment, talc should be applied to alung surface at a dose of 0.05 μg/mm²-10 μg/mm² of surface area coated.

Utilizing silk as an exemplary fibrosis-inducing agent, the total doseof silk delivered from a pulmonary sealant, or coated onto the surfaceof a lung, should not exceed 100 mg (range of 1 μg to 100 mg). In oneembodiment, the total amount of silk released from the sealant should bein the range of 10 μg to 50 mg. The dose per unit area (i.e., the dosageof silk as a function of the surface area of the lung to which drug isapplied) should fall within the range of 0.05 μg-10 μg per mm² ofsurface area coated. In another embodiment, silk should be applied to alung surface at a dose of 0.05 μg/mm²-10 μg/mm² of surface area coated.As specific (polymeric and non-polymeric) drug delivery vehicles andspecific pulmonary sealants can release silk at differing rates, theabove dosing parameters should be utilized in combination with therelease rate of the drug from the sealant such that a minimumconcentration of 0.01 nM to 1000 μM of silk is delivered to the tissue.

Utilizing chitosan as an exemplary fibrosis-inducing agent, the totaldose of chitosan delivered from a pulmonary sealant, or coated onto thesurface of a lung, should not exceed 100 mg (range of 1 μg to 100 mg).In one embodiment, the total amount of chitosan released from thesealant should be in the range of 10 μg to 50 mg. The dose per unit area(i.e., the dosage of chitosan as a function of the surface area of thelung to which drug is applied) should fall within the range of 0.05μg-10 μg per mm² of surface area coated. In another embodiment, chitosanshould be applied to a lung surface at a dose of 0.05 μg/mm²-10 μg/mm²of surface area coated. As specific (polymeric and non-polymeric) drugdelivery vehicles and specific pulmonary sealants can release chitosanat differing rates, the above dosing parameters should be utilized incombination with the release rate of the drug from the sealant such thata minimum concentration of 0.01 nM to 1000 μM of chitosan is deliveredto the tissue.

Utilizing polylysine as an exemplary fibrosis-inducing agent, the totaldose of polylysine delivered from a pulmonary sealant, or coated ontothe surface of a lung, should not exceed 100 mg (range of 1 μg to 100mg). In one embodiment, the total amount of polylysine released from thesealant should be in the range of 10 μg to 50 mg. The dose per unit area(i.e., the dosage of polylysine as a function of the surface area of thelung to which drug is applied) should fall within the range of 0.05μg-10 μg per mm² of surface area coated. In another embodiment,polylysine should be applied to a lung surface at a dose of 0.05μg/mm²-10 μg/mm² of surface area coated. As specific (polymeric andnon-polymeric) drug delivery vehicles and specific pulmonary sealantscan release polylysine at differing rates, the above dosing parametersshould be utilized in combination with the release rate of the drug fromthe pulmonary sealant such that a minimum concentration of 0.01 nM to1000 μM of polylysine is delivered to the tissue.

Utilizing fibronectin as an exemplary fibrosis-inducing agent, the totaldose of fibronectin delivered from a pulmonary sealant, or coated ontothe surface of a lung, should not exceed 100 mg (range of 1 μg to 100mg). In one embodiment, the total amount of fibronectin released fromthe sealant should be in the range of 10 μg to 50 mg. The dose per unitarea (i.e., the dosage of fibronectin as a function of the surface areaof the lung to which drug is applied) should fall within the range of0.05 μg-10 μg per mm² of surface area coated. In another embodiment,fibronectin should be applied to a lung surface at a dose of 0.05μg/mm²-10 μg/mm² of surface area coated. As specific (polymeric andnon-polymeric) drug delivery vehicles and specific pulmonary sealantscan release fibronectin at differing rates, the above dosing parametersshould be utilized in combination with the release rate of the drug fromthe sealant such that a minimum concentration of 0.01 nM to 1000 μM offibronectin is delivered to the tissue.

Utilizing bleomycin as an exemplary fibrosis-inducing agent, the totaldose of bleomycin delivered from a pulmonary sealant, or coated onto thesurface of a lung, should not exceed 100 mg (range of 0.01 μg to 100mg). In one embodiment, the total amount of bleomycin released from thesealant should be in the range of 0.010 μg to 50 mg. The dose per unitarea (i.e., the dosage of bleomycin as a function of the surface area ofthe lung to which drug is applied) should fall within the range of 0.005μg-10 mg per mm² of surface area coated. In another embodiment,bleomycin should be applied to a lung surface at a dose of 0.005μg/mm²-10 μg/mm² of surface area coated. As specific (polymeric andnon-polymeric) drug delivery vehicles and specific pulmonary sealantscan release bleomycin at differing rates, the above dosing parametersshould be utilized in combination with the release rate of the drug fromthe sealant such that a minimum concentration of 0.001 nM to 1000 μM ofbleomycin is delivered to the tissue.

Utilizing CTGF as an exemplary fibrosis-inducing agent, the total doseof CTGF delivered from a pulmonary sealant, or coated onto the surfaceof a lung, should not exceed 100 mg (range of 0.01 μg to 100 mg). In oneembodiment, the total amount of CTGF released from the sealant should bein the range of 0.10 μg to 50 mg. The dose per unit area (i.e., thedosage of CTGF as a function of the surface area of the lung to whichdrug is applied) should fall within the range of 0.005 μg-10 μg per mm²of surface area coated. In another embodiment, CTGF should be applied toa lung surface at a dose of 0.005 μg/mm²-10 μg/mm² of surface areacoated. As specific (polymeric and non-polymeric) drug delivery vehiclesand specific pulmonary sealants can release CTGF at differing rates, theabove dosing parameters should be utilized in combination with therelease rate of the drug from the sealant such that a minimumconcentration of 0.001 nM to 1000 μM of CTGF is delivered to the tissue.

The fibrosing agent (e.g., talc, silk, chitosan, polylysine,fibronectin, bleomycin, CTGF) may be released from the pulmonary sealantsuch that fibrosis in the tissue is promoted for a period ranging fromseveral hours to several months. For example, the fibrosing agent may bereleased in effective concentrations for a period ranging from 1 hour-30days. It should be readily evident given the discussions provided hereinthat analogues and derivatives of the fibrosing agent (e.g., analoguesand derivatives of talc, silk, chitosan, polylysine, fibronectin,bleomycin, CTGF, as previously described) with similar functionalactivity can be utilized for the purposes of this invention; the abovedosing parameters are then adjusted according to the relative potency ofthe analogue or derivative as compared to the parent compound (e.g., acompound twice as potent as the agent is administered at half the aboveparameters, a compound half as potent as the agent is administered attwice the above parameters, etc.).

Optionally, the sealant may alone or additionally comprise aninflammatory cytokine (e.g., TGFβ, PDGF, VEGF, bFGF, TNFα, NGF, GM-CSF,IGF-a, IL-1, IL-1-β, IL-8, IL-6, and growth hormone) or an analogue orderivative thereof. Inflammatory cytokines are to be used informulations at concentrations that range from 0.0001 μg/mL toapproximately 20 mg/mL depending on the specific clinical application,formulation type (e.g., gel, liquid, solid, semi-solid), formulationchemistry, duration of required application, type of medical deviceinterface, and formulation volume and or surface area coverage required.Preferably, the inflammatory cytokine is released in effectiveconcentrations for a period ranging from 1-180 days. The total dose fora single application is typically not to exceed 500 mg (range of 0.0001μg to 100 mg); preferred 0.001 μg to 50 mg. When used as a devicecoating, the dose is per unit area of 0.0001 μg-500 μg per mm²; with apreferred dose of 0.001 μg/mm²-200 μg/mm². Minimum concentration of10⁻¹⁰-10⁻⁴ g/mL of inflammatory cytokine is to be maintained on thedevice surface.

Furthermore, the sealant may alone or additionally comprise an agentthat stimulates cellular proliferation. Examples include: dexamethasone,isotretinoin (13-cis retinoic acid), 17-β-estradiol, estradiol, 1-α-25dihydroxyvitamin D₃, diethylstibesterol, cyclosporine A, L-NAME,all-trans retinoic acid (ATRA), and analogues and derivatives thereof.Doses used are those concentrations which are demonstrated to stimulatecell proliferation. The proliferative agents are to be used informulations at concentrations that range from 0.0000001 to 25 mg/mLdepending on the specific clinical application, formulation type (e.g.,gel, liquid, solid, semi-solid), formulation chemistry, duration ofrequired application, type of medical device interface and formulationvolume and or surface area coverage required. Preferably, theproliferative agent is released in effective concentrations for a periodranging from 1-180 days. The total dose for a single application istypically not to exceed 500 mg (range of 0.0001 μg to 200 mg); preferred0.001 μg to 100 mg. When used as a device coating, the dose is per unitarea of 0.00001 μg-500 μg per mm²; with a preferred dose of 0.0001μg/mm²-200 μg/mm². Minimum concentration of 10⁻¹¹-10⁻⁶ M ofproliferative agent is to be maintained on the device surface.

B. Biologically Active Agent Delivery

The multifunctional compounds may also be used for localized delivery ofvarious drugs and other biologically active agents. Biologically activeagents such as growth factors may be delivered from the composition to alocal tissue site in order to facilitate tissue healing andregeneration. Thus, another embodiment of the invention is a method fordelivering a biologically active agent, where the composition alsoincludes the biologically active agent to be delivered, and steps (a)and (b) are as described for the method of sealing tissue. Step (c)would involve allowing a three-dimensional matrix to form and deliveringthe biologically active agent.

The biologically active agent can either be admixed with thecompositions of the invention or be chemically coupled to themultifunctional compounds in the composition, e.g., by attachment to oneof the reactive groups. For example, processes for covalently bindingbiologically active agents such as growth factors using functionallyactivated polyethylene glycols are described in U.S. Pat. No. 5,162,430to Rhee et al. Such compositions preferably include linkages that can beeasily biodegraded, for example as a result of enzymatic degradation,resulting in the release of the active agent into the target tissue,where it will exert its desired therapeutic effect.

In certain aspects, the biologically active agent may be incorporatedwith a polymeric or non-polymeric carrier to facilitate theincorporation of the agent into the composition. In certain aspects, thecarrier may facilitate sustained release of the agent from thecomposition over a prolonged period of time (e.g., over the course ofseveral days, weeks, or months). For many embodiments, localizeddelivery as well as localized sustained delivery of the agent may bedesired. For example, a therapeutic agent may be admixed with, blendedwith, conjugated to, or, otherwise modified to contain a polymericcomposition (which may be either biodegradable or non-biodegradable) ornon-polymeric composition in order to release the therapeutic agent overa period of time.

Representative examples of biodegradable polymers suitable for thedelivery of therapeutic agents include albumin, collagen, gelatin,hyaluronic acid, starch, cellulose and cellulose derivatives (e.g.,regenerated cellulose, methylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetatephthalate, cellulose acetate succinate, hydroxypropylmethylcellulosephthalate), casein, dextrans, polysaccharides, fibrinogen, poly(etherester) multiblock copolymers, based on poly(ethylene glycol) andpoly(butylene terephthalate), tyrosine-derived polycarbonates (e.g.,U.S. Pat. No. 6,120,491), poly(hydroxyl acids), poly(D,L-lactide),poly(D,L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate),polydioxanone, poly(alkylcarbonate) and poly(orthoesters), polyesters,poly(hydroxyvaleric acid), polydioxanone, polyesters, poly(malic acid),poly(tartronic acid), poly(acrylamides), polyanhydrides,polyphosphazenes, poly(amino acids), poly(alkylene oxide)-poly(ester)block copolymers (e.g., X—Y, X—Y—X, Y—X—Y, R—(Y—X)_(n), or R—(X—Y)_(n),where X is a polyalkylene oxide (e.g., poly(ethylene glycol,poly(propylene glycol) and block copolymers of poly(ethylene oxide) andpoly(propylene oxide) (e.g., PLURONIC® and PLURONIC® R series ofpolymers from BASF Corporation, Mount Olive, N.J.) and Y is a polyester,where the polyester may comprise the residues of one or more of themonomers selected from lactide, lactic acid, glycolide, glycolic acid,ε-caprolactone, γ-caprolactone, hydroxyvaleric acid, hydroxybutyricacid, β-butyrolactone, γ-butyrolactone, γ-valerolactone,γ-decanolactone, δ-decanolactone, trimethylene carbonate,1,4-dioxane-2-one or 1,5-dioxepan-2one (e.g., PLGA, PLA, PCL,polydioxanone and copolymers thereof) and R is a multifunctionalinitiator), and the copolymers as well as blends thereof (see generally,Illum, L., Davids, S. S. (eds.) Polymers in Controlled Drug DeliveryWright, Bristol (1987); Arshady, J. Controlled Release 17:1-22 (1991);Pitt, Int. J. Phar. 59:173-196 (1990); Holland et al., J. ControlledRelease 4:155-0180 (1986)).

Representative examples of non-degradable polymers suitable for thedelivery of therapeutic agents include poly(ethylene-co-vinyl acetate)(“EVA”) copolymers, aromatic polyesters, such as poly(ethyleneterephthalate), silicone rubber, acrylic polymers (polyacrylate,polyacrylic acid, polymethylacrylic acid, polymethylmethacrylate,poly(butyl methacrylate)), poly(alkylcynoacrylate) (e.g.,poly(ethylcyanoacrylate), poly(butylcyanoacrylate)poly(hexylcyanoacrylate) poly(octylcyanoacrylate)), acrylic resin,polyethylene, polypropylene, polyamides (nylon 6,6), polyurethanes(e.g., CHRONOFLEX® AL and CHRONOFLEX AR® (both from CardioTechInternational, Inc., Woburn, Mass.), TECOFLEX®, and BIONATE® (PolymerTechnology Group, Inc., Emeryville, Calif.)), poly(ester urethanes),poly(ether urethanes), poly(ester-urea), polyethers (poly(ethyleneoxide), poly(propylene oxide), polyoxyalkylene ether block copolymersbased on ethylene oxide and propylene oxide such as the PLURONIC®polymers (e.g., F-127 or F87) from BASF Corporation (Mount Olive, N.J.),and poly(tetramethylene glycol), styrene-based polymers (polystyrene,poly(styrene sulfonic acid),poly(styrene)-block-poly(isobutylene)-block-poly(styrene),poly(styrene)-poly(isoprene) block copolymers), and vinyl polymers(polyvinylpyrrolidone, poly(vinyl alcohol), poly(vinyl acetatephthalate) as well as copolymers and blends thereof. Polymers may alsobe developed which are either anionic (e.g., alginate, carrageenan,carboxymethyl cellulose, poly(acrylamido-2-methyl propane sulfonic acid)and copolymers thereof, poly(methacrylic acid and copolymers thereof andpoly(acrylic acid) and copolymers thereof, as well as blends thereof, orcationic (e.g., chitosan, poly-L-lysine, polyethylenimine, andpoly(allyl amine)) and blends thereof (see generally, Dunn et al., J.Applied Polymer Sci. 50:353-365 (1993); Cascone et al., J. MaterialsSci.: Materials in Medicine 5:770-774 (1994); Shiraishi et al., Biol.Pharm. Bull. 16(11):1164-1168 (1993); Thacharodi and Rao, Intl J. Pharm.120:115-118 (1995); Miyazaki et al., Intl J. Pharm. 118:257-263 (1995).

Some examples of preferred polymeric carriers for the practice of thisinvention include poly(ethylene-co-vinyl acetate), polyurethanes, poly(D,L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomersand polymers, poly (glycolic acid), copolymers of lactic acid andglycolic acid, copolymers of lactide and glycolide, poly (caprolactone),poly (valerolactone), polyanhydrides, copolymers of poly (caprolactone)or poly (lactic acid) with a polyethylene glycol (e.g., MePEG), blockcopolymers of the form X—Y, X—Y—X, Y—X—Y, R—(Y—X)_(n), or R—(X—Y)_(n),[where X is a polyalkylene oxide (e.g., poly(ethylene glycol,poly(propylene glycol) and block copolymers of poly(ethylene oxide) andpoly(propylene oxide) (e.g., PLURONIC® and PLURONIC® R series ofpolymers from BASF Corporation, Mount Olive, N.J.)) and Y is apolyester, where the polyester may comprise the residues of one or moreof the monomers selected from lactide, lactic acid, glycolide, glycolicacid, ε-caprolactone, γ-caprolactone, hydroxyvaleric acid,hydroxybutyric acid, β-butyrolactone, γ-butyrolactone, γ-valerolactone,γ-decanolactone, δ-decanolactone, trimethylene carbonate,1,4-dioxane-2-one or 1,5-dioxepan-2-one; R is a multifunctionalinitiator; and n is 2 to 12], silicone rubbers,poly(styrene)block-poly(isobutylene)-block-poly(styrene), poly(acrylate)polymers and blends, admixtures, or co-polymers of any of the above.Other preferred polymers include collagen, poly(alkylene oxide)-basedpolymers, polysaccharides such as hyaluronic acid, chitosan and fucans,and copolymers of polysaccharides with degradable polymers.

Other representative polymers capable of sustained localized delivery oftherapeutic agents described herein include carboxylic polymers,polyacetates, polycarbonates, polyethers, polyethylenes,polyvinylbutyrals, polysilanes, polyureas, polyoxides, polystyrenes,polysulfides, polysulfones, polysulfonides, polyvinylhalides,pyrrolidones, rubbers, thermal-setting polymers, cross-linkable acrylicand methacrylic polymers, ethylene acrylic acid copolymers, styreneacrylic copolymers, vinyl acetate polymers and copolymers, vinyl acetalpolymers and copolymers, epoxies, melamines, other amino resins,phenolic polymers, and copolymers thereof, water-insoluble celluloseester polymers (including cellulose acetate propionate, celluloseacetate, cellulose acetate butyrate, cellulose nitrate, celluloseacetate phthalate, and mixtures thereof), polyvinylpyrrolidone,polyethylene glycols, polyethylene oxide, polyvinyl alcohol, polyethers,polysaccharides, hydrophilic polyurethane, polyhydroxyacrylate, dextran,xanthan, hydroxypropyl cellulose, and homopolymers and copolymers ofN-vinylpyrrolidone, N-vinyllactam, N-vinyl butyrolactam, N-vinylcaprolactam, other vinyl compounds having polar pendant groups, acrylateand methacrylate having hydrophilic esterifying groups, hydroxyacrylate,and acrylic acid, and combinations thereof; cellulose esters and ethers,ethyl cellulose, hydroxyethyl cellulose, cellulose nitrate, celluloseacetate, cellulose acetate butyrate, cellulose acetate propionate,natural and synthetic elastomers, rubber, acetal, styrene polybutadiene,acrylic resin, polyvinylidene chloride, polycarbonate, homopolymers andcopolymers of vinyl compounds, polyvinylchloride, and polyvinylchlorideacetate.

Representative examples of patents relating to drug-delivery polymersand their preparation include PCT Publication Nos. WO 98/19713, WO01/17575, WO 01/41821, WO 01/41822, and WO 01/15526 (as well as thecorresponding U.S. applications), U.S. Pat. Nos. 4,500,676; 4,582,865;4,629,623; 4,636,524; 4,713,448; 4,795,741; 4,913,743; 5,069,899;5,099,013; 5,128,326; 5,143,724; 5,153,174; 5,246,698; 5,266,563;5,399,351; 5,525,348; 5,800,412; 5,837,226; 5,942,555; 5,997,517;6,007,833; 6,071,447; 6,090,995; 6,106,473; 6,110,483; 6,121,027;6,156,345; 6,214,901; 6,368,611; 6,630,155; 6,528,080; RE37,950;6,461,631; 6,143,314; 5,990,194; 5,792,469; 5,780,044; 5,759,563;5,744,153; 5,739,176; 5,733,950; 5,681,873; 5,599,552; 5,340,849;5,278,202; 5,278,201; 6,589,549; 6,287,588; 6,201,072; 6,117,949;6,004,573; 5,702,717; 6,413,539; 5,714,159; 5,612,052; and U.S. PatentApplication Publication Nos. 2003/0068377, 2002/0192286, 2002/0076441,and 2002/0090398.

It should be obvious to one of skill in the art that the polymers asdescribed herein can also be blended or copolymerized in variouscompositions as required to deliver therapeutic doses of biologicallyactive agents.

Drug delivery vehicles may take a variety of forms. For example, thecarrier may be in the form of microspheres (e.g., PLGA, PLLA, PDLLA,PCL, gelatin, polydioxanone, poly(alkylcyanoacrylate)), nanospheres(PLGA, PLLA, PDLLA, PCL, gelatin, polydioxanone,poly(alkylcyanoacrylate)) (see, e.g., Hagan et al., Proc. Intern. Symp.Control Rel. Bioact. Mater. 22 (1995); Kwon et al., Pharm Res.12(2):192-195; Kwon et al., Pharm Res. 10(7):970-974; Yokoyama et al.,J. Contr. Rel. 32:269-277 (1994); Gref et al., Science 263:1600-1603(1994); Bazile et al., J. Pharm. Sci. 84:493-498 (1994), emulsions (see,e.g., Tan et al., Pharm Res. 4:62-165 (1987), microemulsions, micelles(SDS, block copolymers of the form X—Y, Y—X—Y, R—(Y—X)_(n), R—(X—Y)_(n)and X—Y—X (where X in a polyalkylene oxide (e.g., poly(ethylene glycol,poly(propylene glycol) and block copolymers of poly(ethylene oxide) andpoly(propylene oxide) (e.g., PLURONIC® and PLURONIC® R series ofpolymers from BASF Corporation, Mount Olive, N.J.) and Y is abiodegradable polyester, where the polyester may comprise the residuesof one or more of the monomers selected from lactide, lactic acid,glycolide, glycolic acid, ε-caprolactone, γ-caprolactone, hydroxyvalericacid, hydroxybutyric acid, β-butyrolactone, γ-butyrolactone,γ-valerolactone, γ-decanolactone, δ-decanolactone, trimethylenecarbonate, 1,4-dioxane-2-one or 1,5-dioxepan-2one (e.g., PLG-PEG-PLG)and R is a multifunctional initiator), and zeolites.

Other types of carriers that may utilized to contain and delivertherapeutic agents described herein include: cyclodextrins, such ashydroxypropyl cyclodextrin (Cserhati and Hollo, Int. J. Pharm. 108:69-75(1994)), liposomes (see, e.g., Sharma et al., Cancer Res. 53:5877-5881,1993; Sharma and Straubinger, Pharm. Res. 11(60):889-896, 1994; WO93/18751; U.S. Pat. No. 5,242,073), liposome/gel (WO 94/26254),nanocapsules (Bartoli et al., J. Microencapsulation 7(2):191-197, 1990),implants (Jampel et al., Invest. Ophthalm. Vis. Science34(11):3076-3083, 1993; Walter et al., Cancer Res. 54:22017-2212, 1994),nanoparticles (Violante and Lanzafame PAACR), nanoparticles—modified(U.S. Pat. No. 5,145,684), nanoparticles (surface modified) (U.S. Pat.No. 5,399,363), micelles such as are described in Alkan-Onyuksel et al.,Pharm. Res. 11(2):206-212, 1994), micelle (surfactant) (U.S. Pat. No.5,403,858), synthetic phospholipid compounds (U.S. Pat. No. 4,534,899),gas borne dispersion (U.S. Pat. No. 5,301,664), liquid emulsions, foam,spray, gel, lotion, cream, ointment, dispersed vesicles, particles ordroplets solid- or liquid-aerosols, microemulsions (U.S. Pat. No.5,330,756), polymeric shell (nano- and micro-capsule) (U.S. Pat. No.5,439,686), and implants (U.S. Pat. No. 4,882,168).

Within certain aspects of the present invention, therapeutic agents maybe fashioned in the form of microspheres, microparticles, and/ornanoparticles having any size ranging from 50 nm to 500 lam, dependingupon the particular use. These compositions can be formed byspray-drying methods, milling methods, coacervation methods, w/oemulsion methods, w/o/w emulsion methods, and solvent evaporationmethods. In other aspects, these compositions can includemicroemulsions, emulsions, liposomes and micelles. Compositionscomprising a drug-loaded carrier may also be readily applied as a“spray”, which solidifies into a film or coating for use as adevice/implant surface coating or to line the tissues of theimplantation site. Such sprays may be prepared from microspheres of awide array of sizes, including for example, from 0.1 μm to 3 μm, from 10μm to 30 μm, and from 30 μm to 100 μm.

In one aspect, biologically active agents such as growth factors orfibrosis-inducing agents may be delivered from the composition to alocal tissue site in order to facilitate scar formation, tissue healing,and/or regeneration. Thus, in one aspect, a method is provided fordelivering a biologically active agent, where the composition alsoincludes the biologically active agent (e.g., a fibrosing agent) to bedelivered, and steps (a) and (b) are as described for the method ofsealing tissue. Step (c) would involve allowing a three-dimensionalmatrix to form and delivering the biologically active agent.

As described above, the composition may include an agent that promotesfibrosis. Compositions that include a fibrosis-inducing agent may beused in a variety of applications, including, without limitation, tissueaugmentation, bone growth, treatment of aneurysms, filling and blockingof voids in the body, medical devices coatings, and for use in sealantcompositions.

In certain embodiments, the fibrosis or adhesion-inducing agent is silk.Silk refers to a fibrous protein, and may be obtained from a number ofsources, typically spiders and silkworms. Typical silks contain about75% of actual fiber, referred to as fibroin, and about 35% sericin,which is a gummy protein that holds the filaments together. Silkfilaments are generally very fine and long—as much as 300-900 meterslong. There are several species of domesticated silkworm that are usedin commercial silk production, however, Bombyx mori is the most common,and most silk comes from this source. Other suitable silkworms includePhilosamia cynthia ricini, Antheraea yamamai, Antheraea pernyi, andAntheraea mylitta. Spider silk is relatively more difficult to obtain,however, recombinant techniques hold promise as a means to obtain spidersilk at economical prices (see, e.g., U.S. Pat. Nos. 6,268,169;5,994,099; 5,989,894; and 5,728,810, which are exemplary only).Biotechnology has allowed researchers to develop other sources for silkproduction, including animals (e.g., goats) and vegetables (e.g.,potatoes). Silk from any of these sources may be used in the presentinvention.

A commercially available silk protein is available from Croda, Inc., ofParsippany, N.J., and is sold under the trade names CROSILK LIQUID (silkamino acids), CROSILK 10,000 (hydrolyzed silk), CROSILK POWDER (powderedsilk), and CROSILKQUAT (cocodiammonium hydroxypropyl silk amino acid).Another example of a commercially available silk protein is SERICIN,available from Pentapharm, LTD, a division of Kordia, BV, of theNetherlands. Further details of such silk protein mixtures can be foundin U.S. Pat. No. 4,906,460, to Kim, et al., assigned to Sorenco. Silkuseful in the present invention includes natural (raw) silk, hydrolyzedsilk, and modified silk, i.e., silk that has undergone a chemical,mechanical, or vapor treatment, e.g., acid treatment or acylation (see,e.g., U.S. Pat. No. 5,747,015).

Raw silk is typically twisted into a strand sufficiently strong forweaving or knitting. Four different types of silk thread may be producedby this procedure: organzine, crepe, tram, and thrown singles. Organzineis a thread made by giving the raw silk a preliminary twist in onedirection and then twisting two of these threads together in theopposite direction. Crepe is similar to organzine but is twisted to amuch greater extent. Twisting in only one direction two or more raw silkthreads makes tram. Thrown singles are individual raw silk threads thatare twisted in only one direction. Any of these types of silk threadsmay be used in the present invention.

The silk used in the present invention may be in any suitable form thatallows the silk to be joined with the medical implant, e.g., the silkmay be in thread or powder-based forms. The silk can be prepared in thepowdered form by several different methods. For example the silk can bemilled (e.g., cryomill) into a powdered form. Alternatively the silk canbe dissolved in a suitable solvent (e.g., HFIP or 9M LiBr) and thensprayed (electrospray, spray dry) or added to a non-solvent to produce apowder. Furthermore, the silk may have any molecular weight, wherevarious molecular weights are typically obtained by the hydrolysis ofnatural silk, where the extent and harshness of the hydrolysisconditions determines the product molecular weight. For example, thesilk may have an average (number or weight) molecular weight of about200 to 5,000. See, e.g., JP-B-59-29199 (examined Japanese patentpublication) for a description of conditions that may be used tohydrolyze silk.

A discussion of silk may be found in the following documents, which areexemplary only: Hinman, M. B. et al. “Synthetic spider silk: a modularfibre” Trends in Biotechnology 18(9) 374-379 (2000); Vollrath, F. andKnight, D. P. “Liquid crystalline spinning of spider silk” Nature410(6828) 541-548 (2001); and Hayashi, C. Y. et al. “Hypotheses thatcorrelate the sequence, structure, and mechanical properties of spidersilk proteins” Int. J. Biol. Macromolecules 24(2-3), 265-270 (1999); andU.S. Pat. No. 6,427,933.

Other representative examples of fibrosis and adhesion-inducing agentsinclude irritants (e.g., talc, talcum powder, copper, metallic beryllium(or its oxides), wool (e.g., animal wool, wood wool, and syntheticwool), quartz dust, silica, crystalline silicates), polymers (e.g.,polylysine, polyurethanes, poly(ethylene terephthalate),polytetrafluoroethylene (PTFE), poly(alkylcyanoacrylates), andpoly(ethylene-co-vinylacetate)); vinyl chloride and polymers of vinylchloride; peptides with high lysine content; growth factors andinflammatory cytokines involved in angiogenesis, fibroblast migration,fibroblast proliferation, ECM synthesis and tissue remodeling, such asepidermal growth factor (EGF) family, transforming growth factor-alpha(TGF-α), transforming growth factor-beta (TGF-β-1, TGF-β-2, TGF-β-3),platelet-derived growth factor (PDGF), fibroblast growth factor(acidic—aFGF; and basic—bFGF), fibroblast stimulating factor-1,activins, vascular endothelial growth factor (including VEGF-2, VEGF-3,VEGF-A, VEGF-B, VEGF-C, placental growth factor—PIGF), angiopoietins,insulin-like growth factors (IGF), hepatocyte growth factor (HGF),connective tissue growth factor (CTGF), myeloid colony-stimulatingfactors (CSFs), monocyte chemotactic protein, granulocyte-macrophagecolony-stimulating factors (GM-CSF), granulocyte colony-stimulatingfactor (G-CSF), macrophage colony-stimulating factor (M-CSF),erythropoietin, interleukins (particularly IL-1, IL-8, and IL-6), tumornecrosis factor-alpha (TNF-α), nerve growth factor (NGF),interferon-α-interferon-β-histamine, endothelin-1, angiotensin II,growth hormone (GH), and synthetic peptides, analogues or derivatives ofthese factors are also suitable for release from specific implants anddevices to be described later. Other examples include CTGF (connectivetissue growth factor); inflammatory microcrystals (e.g., crystallineminerals such as crystalline silicates); bromocriptine, methylsergide,methotrexate, chitosan, N-carboxybutyl chitosan, carbon tetrachloride,thioacetamide, fibrosin, ethanol, bleomycin, naturally occurring orsynthetic peptides containing the Arg-Gly-Asp (RGD) sequence, generallyat one or both termini (see e.g., U.S. Pat. No. 5,997,895), and tissueadhesives, such as cyanoacrylate and crosslinked poly(ethyleneglycol)-methylated collagen compositions, such as described below.

Other examples of fibrosis-inducing agents include agents that promotebone growth, such as for example bone morphogenic proteins. Examples ofbone morphogenic proteins include the following: BMP-2, BMP-3, BMP-4,BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11,BMP-12, BMP-13, BMP-14, BMP-15, and BMP-16. Of these, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, and BMP-7. Bone morphogenic proteins are described,for example, in U.S. Pat. Nos. 4,877,864; 5,013,649; 5,661,007;5,688,678; 6,177,406; 6,432,919; and 6,534,268, and Wozney, J. M. etal., Science 242(4885):1528-1534 (1988).

Other representative examples of fibrosis-inducing agents includecomponents of extracellular matrix (e.g., fibronectin, fibrin,fibrinogen, collagen (e.g., bovine collagen), fibrillar andnon-fibrillar collagen, adhesive glycoproteins, proteoglycans (e.g.,heparin sulfate, chondroitin sulfate, dermatan sulfate), hyaluronan,secreted protein acidic and rich in cysteine (SPARC), thrombospondins,tenacin, and cell adhesion molecules (including integrins, vitronectin,fibronectin, laminin, hyaluronic acid, elastin, bitronectin), proteinsfound in basement membranes, and fibrosin) and inhibitors of matrixmetalloproteinases, such as TIMPs (tissue inhibitors of matrixmetalloproteinases) and synthetic TIMPs, e.g., marimistat, batimistat,doxycycline, tetracycline, minocycline, TROCADE, Ro-1130830, CGS 27023A,and BMS-275291.

Within various embodiments of the invention, a composition incorporatesa compound which acts to stimulate cellular proliferation. In certainembodiments, a composition may incorporate a compound which acts tostimulate cellular proliferation in addition to a fibrosing agent.Representative examples of agents that stimulate cellular proliferationinclude, pyruvic acid, naltrexone, leptin, D-glucose, insulin,amlodipine, alginate oligosaccharides, minoxidil, dexamethasone,isotretinoin (13-cis retinoic acid), 17-β-estradiol, estradiol, 1-α-25dihydroxyvitamin D₃, diethylstibesterol, cyclosporine A, L-NAME(L-NG-nitroarginine methyl ester (hydrochloride)), all-trans retinoicacid (ATRA), and analogues and derivatives thereof. Other examples ofagents that stimulate cellular proliferation include: sphingosine1-phosphate receptor agonist (e.g., FTY-720 (1,3-propanediol,2-amino-2-(2-(4-octylphenyl)ethyl)-, hydrochloride; immunostimulants,such as Imupedone (methanone,[5-amino-2-(4-methyl-1-piperidinyl)phenyl](4-chlorophenyl)-, DIAPEP227synthetic peptide (Peptor Ltd., Israel)); and nerve growth factoragonist, e.g., NG-012(5H,9H,13H,21H,25H,-dibenzo[k,u][1,5,9,15,19]pentaoxacyclotetracosin-5,9,13,21,25-pentone,7,8,11,12,15,16,23,24,27,28-decahydro-2,4,18,20-tetrahydroxy-1′-(hydroxymethyl)-7,15,23,27-tetramethyl-,NG-121, SS-701 (2,2′:6′,2″-terpyridine, 4′-(4-methylphenyl)-,trihydrochloride, AMPAlex (piperidine, 1-(6-quinoxalinylcarbonyl)-,RGH-2716(8-[4,4-bis(4-fluorophenyl)butyl]-3-(1,1-dimethylethyl)-4-methylene-1-oxa-3,8-diaza-spiro[4.5]decan-2-one,and TDN-345 (1-oxa-3,8-diazaspiro[4.5]decan-2-one,8-[4,4-bis(4-fluorophenyl)butyl]-3-(1,1-dimethylethyl)-4-methylene-).

Particularly useful biologically active agents for use in thecompositions of the present invention are cytokines, which arebiologically active molecules including growth factors and activepeptides, which aid in healing or regrowth of normal tissue. Thefunction of cytokines is two-fold: 1) they can incite local cells toproduce new collagen or tissue, or 2) they can attract cells to the sitein need of correction. As such, cytokines, as well as appropriatecombinations of cytokines, serve to encourage “biological anchoring” ofan implant within the host tissue, by facilitating the regrowth andremodeling of the implant into normal bone tissue. Cytokines may also beused in the treatment of wounds.

Examples of cytokines include, by way of illustration and notlimitation, transforming growth factors (TGFs); fibroblast growthfactors (FGFs), including both acidic FGF and basic FGF; plateletderived growth factors (PDGFs) such as PDGF-AA, PDGF-AB, and PDGF-BB;epidermal growth factors (EGFs); connective tissue activated peptides(CTAPs); colony stimulating factors (CSFs); erythropoietin (EPO); nervegrowth factor (NGF); osteogenic factors; β-thromboglobulin; tumornecrosis factors (TNFs); interleukins; interferons (IFNs); bonemorphogenic protein (BMP); and biologically active analogs, fragments,and derivatives of such growth factors. Members of the transforminggrowth factor (TGF) supergene family, which are multifunctionalregulatory proteins, are particularly preferred. Members of the TGFsupergene family include TGF-α and the transforming growth factors (forexample, TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (forexample, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);heparin-binding growth factors, e.g., FGFs; EGFs; PDGFs; insulin-likegrowth factors (IGFs); inhibins such as Inhibin A and Inhibin B; growthdifferentiating factors, e.g., GDF-1); and activins such as Activin A,Activin B, Activin AB. Growth factors can be isolated from native ornatural sources, such as from mammalian cells, or can be preparedsynthetically, such as by recombinant DNA techniques or by variouschemical processes. In addition, analogs, fragments, or derivatives ofthese factors can be used, provided that they exhibit at least some ofthe biological activity of the native molecule. For example, analogs canbe prepared by expression of genes altered by site-specific mutagenesisor other genetic engineering techniques.

By varying the relative molar amounts of the different the reactivegroups on the multifunctional compounds, it is possible to alter the netcharge of the resulting three-dimensional matrix, in order to prepare amatrix for the delivery of a charged compound such as a protein orionizable drug. As such, the delivery of charged proteins or drugs,which would normally diffuse rapidly out of a neutral carrier matrix,can be controlled.

For example, if a molar excess of nucleophilic groups are used, theresulting matrix has a net positive charge and can be used to ionicallybind and deliver negatively charged compounds. Similarly, if a molarexcess of electrophilic groups are used, the resulting matrix has a netnegative charge and can be used to ionically bind and deliver positivelycharged compounds. Examples of negatively and positively chargedcompounds that can be delivered from these matrices include variousdrugs, cells, proteins, and polysaccharides. Negatively chargedcollagens, such as succinylated collagen, and glycosaminoglycanderivatives such as sodium hyaluronate, keratan sulfate, keratosulfate,sodium chondroitin sulfate A, sodium dermatan sulfate B, sodiumchondroitin sulfate C, heparin, esterified chondroitin sulfate C, andesterified heparin, can also be effectively incorporated into the matrixas described above. Positively charged collagens, such as methylatedcollagen, and glycosaminoglycan derivatives such as esterifieddeacetylated hyaluronic acid, esterified deacetylated desulfatedchondroitin sulfate A, esterified deacetylated desulfated chondroitinsulfate C, deacetylated desulfated keratan sulfate, deacetylateddesulfated keratosulfate, esterified desulfated heparin, and chitosan,can also be similarly incorporated.

In another aspect, biologically active agents such asfibrosis-inhibiting agents may be delivered from the composition to alocal tissue site in order to inhibit scar formation, tissue healing,and/or regeneration. Thus, in one aspect, a method is provided fordelivering a biologically active agent, where the composition alsoincludes the biologically active agent (e.g., a fibrosis-inhibitingagent) to be delivered, and steps (a) and (b) are as described for themethod of sealing tissue. Step (c) would involve allowing athree-dimensional matrix to form to deliver the biologically activeagent. Compositions that include a fibrosis-inhibiting agent may be usedin a variety of applications, including, without limitation, surgicaladhesion prevention and medical devices coatings. Numerous therapeuticcompounds have been identified that are of utility in the inventionincluding:

Angiogenesis Inhibitors

In one embodiment, the pharmacologically active compound inhibitsangiogenesis (i.e., angiogenesis inhibitor), such as, for example, 2-ME(NSC-659853), PI-88 (D-mannose),O-6-O-phosphono-α-D-mannopyranosyl-(1-3)-O-α-D-mannopyranosyl-(1-3)-O-α-D-mannopyranosyl-(1-3)-O-α-D-mannopyranosyl-(1-2)-hydrogensulphate), thalidomide (1H-isoindole-1,3(2H)-dione,2-(2,6-dioxo-3-piperidinyl)-), CDC-394, CC-5079, ENMD-0995(S-3-amino-phthalidoglutarimide), AVE-8062A, vatalanib, SH-268,halofuginone hydrobromide, atiprimod dimaleate(2-azaspivo[4.5]decane-2-propanamine, N,N-diethyl-8,8-dipropyl,dimaleate), ATN-224, CHIR-258, combretastatin A-4 (phenol,2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)ethenyl]-, (Z)—), GCS-100LE, oran analogue or derivative thereof.

Other examples of angiogenesis inhibitors for use in the compositions ofthe invention include: 2-methoxyestradiol, A6, ABT-510, ABX-IL8,actimid, Ad5FGF-4, AG3340, α-5-β-1 integrin antibody, AMG001, anecortaveacetate, angiocol, angiogenix, angiostatin, angiozyme, antiangiogenicantithrombin 3, anti-VEGF, anti-VEGF Mab, aplidine, aptosyn, ATN-161,avastin, AVE8062A, Bay 12-9566, benefin, BioBypass CAD, MS275291, CAI,carboxymidotriazole, CC 4047, CC 5013, CC7085, CDC801, Celebrex,CEP-7055, CGP-41251/PKC412, cilengitide, CM101, col-3, combretastatin,combretastatin A4P, CP-547, 632, CP-564, 959, Del-1, dexrazoxane,didemnin B, DMXAA, EMD 121974, endostatin, FGF (AGENT 3), flavopiridol,GBC-100, genistein concentrated polysaccharide, green tea extract, HIF-1α, human chorio-gonadotrophin, IM862, INGN 201, interferon α-2a,interleukin-12, iressa, ISV-120, LY317615, LY-333531, Mab huJ591-DOTA-90Yttrium, marimastat, Medi-522, metaret, neoretna, neovastat, NM-3, NPe6,NV1FGF, octreotide, oltipraz, paclitaxel, pegaptanib sodium,penicillamine, pentosan polysulphate, prinomastat, PSK, psorvastat,PTK787/ZK222584, ranibizumab, razoxane, replistatatin, revimid, RhuMab,Ro317453, squalamine, SU101, SU11248, SU5416, SU6668, tamoxifen,tecogalan sodium, temptostatin, tetrathiomol, tetrathiomolybdate,thalomid, TNP-470, UCN-01, VEGF, VEGF trap, Vioxx, vitaxin, vitaxin-2,ZD6126, ZD6474, angiostatin (plasminogen fragment), a TIMPs,antiangiogenic antithrombin III, pigment epithelial-derived factor(PEDF), canstatin, placental ribonuclease inhibitor, cartilage-derivedinhibitor (CDI), plasminogen activator inhibitor, CD59 complementfragment, platelet factor-4, endostatin (collagen XVIII fragment),prolactin 16 kD fragment, fibronectin fragment, proliferin-relatedprotein, gro-beta, a retinoid, a heparinase, tetrahydrocortisol-S,heparin hexasaccharide fragment, thrombospondin-1, human chorionicgonadotropin, transforming growth factor-beta, interferon-alpha,interferon-beta, or interferon-gamma, tumistatin, interferon inducibleprotein, vasculostatin, interleukin-12, vasostatin (calreticulinfragment), kringle 5 (plasminogen fragment), angioarrestin, or2-methoxyestradiol. Angiogenesis inhibitors also include antagonists ofangiogenin, placental growth factor, angiopoietin-1, platelet-derivedendothelial cell growth factor, Del-1, platelet-derived growthfactor-BB, aFGF, bFGF, pleiotrophin, follistatin, proliferin,granulocyte colony-stimulating factor, transforming growth factor-alpha,hepatocyte growth factor, transforming growth factor-beta,interleukin-8, tumor necrosis factor-alpha, leptin, vascular endothelialgrowth factor, midkine, progranulin, 2-methoxyestradiol (PANZEM)(EntreMed), A6, ABT-510, ABX-IL8 (Abgenix), actimid, Ad5FGF-4(Collateral Therapeutics), AG3340 (Agouron Pharmaceuticals Inc. LaJolla,Calif.), α-5-β-1 integrin antibody, AMG001 (AnGes/DaichiPharmaceuticals), anecortave acetate (Retaane, Alcon), angiocol,angiogenix (Endovasc Ltd), angiostatin (EntreMed), angiozyme,antiangiogenic antithrombin 3 (Genzyme Molecular Oncology), anti-VEGF(Genentech), anti-VEGF Mab, aplidine, aptosyn, ATN-161, avastin(bevacizumab), AVE8062A, Bay 12-9566 (Bayer Corp. West Haven, Conn.),benefin, BioBypass CAD (VEGF-121) (GenVec), MS275291, CAI (carboxy-amidoimidazole), carboxymidotriazole, CC 4047 (Celgene), CC 5013 (Celgene),CC7085, CDC 801 (Celgene), Celebrex (Celecoxib), CEP-7055,CGP-41251/PKC412, cilengitide, CM101 (Carborned Brentwood, Term.), col-3(CollaGenex Pharmaceuticals Inc. Newton, Pa.), combretastatin,combretastatin A4P (Oxigene/Bristol-Myers Squibb), CP-547, 632, CP-564,959, Del-1 (VLTS-589) (Valentis), dexrazoxane, didemnin B, DMXAA, EMD121974, endostatin (EntreMed), FGF (AGENT 3) (Berlex (Krannert Instituteof Cardiology)), flavopiridol, GBC-100, genistein concentratedpolysaccharide, green tea extract, HIF-1 alpha (Genzyme), humanchorio-gonadotrophin, IM862 (Cytran), INGN 201, interferon-α-2a,interleukin-12, iressa, ISV-120 (Batimastat), LY317615, LY-333531 (EliLilly and Company), Mab huJ591-DOTA-90 Yttrium (90Y), marimastat(British Biotech Inc. Annapolis, Md.), Medi-522, metaret (suramin),neoretna, neovastat (AEtema Laboratories), NM-3, NPe6, NV1FGF(Gencell/Aventis), octreotide, oltipraz, paclitaxel (e.g., taxol,docetaxel, or paxene), pegaptanib sodium (Eyetech), penicillamine,pentosan polysulphate, PI-88, prinomastat (Agouron Pharmaceuticals),PSK, psorvastat, PTK787/ZK222584, ranibizumab (Lucentis, Genentech),razoxane, replistatatin (Platelet factor-4), revimid, RhuMab, Ro317453,squalamine (Magainin Pharmaceuticals, Inc. Plymouth Meeting, Pa.), SU101(Sugen Inc. Redwood City, Calif.), SU11248, SU5416 (Sugen), SU6668(Sugen), tamoxifen, tecogalan sodium, temptostatin, tetrathiomol,tetrathiomolybdate,

Anti-angiogensis compounds found in vivo may be used in the compositionsand methods described including angiostatin (plasminogen fragment),metalloproteinase inhibitors (TIMPs), antiangiogenic antithrombin III(aaATIII), pigment epithelial-derived factor (PEDF), canstatin,placental ribonuclease inhibitor, cartilage-derived inhibitor (CDI),plasminogen activator inhibitor, CD59 complement fragment, plateletfactor-4 (PF4), endostatin (collagen XVIII fragment), prolactin 16 kDfragment, fibronectin fragment, proliferin-related protein, gro-beta,retinoids, heparinases, tetrahydrocortisol-S, heparin hexasaccharidefragment, thrombospondin-1, human chorionic gonadotropin (hCG),transforming growth factor-beta, interferon-alpha/beta/gamma,tumistatin, interferon inducible protein (IP-10), vasculostatin,interleukin-12 (IL-12), vasostatin (calreticulin fragment), kringle 5(plasminogen fragment), angioarrestin, and 2-methoxyestradiol.

Compounds that inhibit, block, or antagonize the angiogenic activity ofthe following species in vivo may be used in the methods andcompositions described herein including angiogenin, placental growthfactor, angiopoietin-1, platelet-derived endothelial cell growth factor(PD-ECGF), Del-1, platelet-derived growth factor-BB (PDGF-BB),fibroblast growth factors: acidic (aFGF) and basic (bFGF), pleiotrophin(PTN), follistatin, proliferin, granulocyte colony-stimulating factor(G-CSF), transforming growth factor-alpha (TGF-α), hepatocyte growthfactor (HGF)/scatter factor (SF), transforming growth factor-beta(TGF-β), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha),leptin, vascular endothelial growth factor (VEGF)/vascular permeabilityfactor (VPF), midkine, and progranulin.

Other examples of angiogenesis inhibitors for use in the presentcompositions include 2-methoxyestradiol, prinomastat, batimastat, BAY12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetic,dimethylxanthenone acetic acid, EMD 121974, endostatin, IM-862,marimastat, matrix metalloproteinase, penicillamine, PTK787/ZK 222584,RPI.4610, squalamine, squalamine lactate, SU5416, (.+−.)-thalidomide,S-thalidomide, R-thalidomide, TNP-470, combretastatin, paclitaxel,tamoxifen, COL-3, neovastat, BMS-275291, SU6668, interferon-alpha,anti-VEGF antibody, Medi-522 (Vitaxin II), CAI, celecoxib,Interleukin-12, IM862, Amilloride, Angiostatin, Protein, AngiostatinK1-3, Angiostatin K1-5, Captopril, DL-alpha-Difluoromethylomithine,DL-alpha-Difluoromethylomithine HCl, His-Tag, Endostatin, Protein,Fumagillin, Herbimycin A, 4-Hydroxyphenylretinamide, gamma-interferon,Juglone, Laminin, Laminin Hexapeptide, Laminin Pentapeptide, LavendustinA, Medroxyprogesterone, Medroxyprogesterone Acetate, Minocycline,Minocycline HCl, Placental Ribonuclease Inhibitor, Suramin, Sodium SaltSuramin, Human Platelet Thrombospondin, Tissue Inhibitor ofMetalloproteinase 1, Neutrophil Granulocyte Tissue Inhibitor ofMetalloproteinase 1, and Rheumatoid Synovial Fibroblast Tissue Inhibitorof Metalloproteinase 2.

5-Lipdxygenase Inhibitors and Antagonists

In another embodiment, the pharmacologically active compound is a5-lipoxygenase inhibitor or antagonist (e.g., Wy-50295(2-naphthaleneacetic acid, alpha-methyl-6-(2-quinolinylmethoxy)-, (S)—),ONO-LP-269 (2,11,14-eicosatrienamide,N-(4-hydroxy-2-(1H-tetrazol-5-yl)-8-quinolinyl)-, (E,Z,Z)—), licofelone(1H-pyrrolizine-5-acetic acid,6-(4-chlorophenyl)-2,3-dihydro-2,2-dimethyl-7-phenyl-), CMI-568 (urea,N-butyl-N-hydroxy-N′-(4-(3-(methylsulfonyl)-2-propoxy-5-(tetrahydro-5-(3,4,5-trimethoxyphenyl)-2-furanyl)phenoxy)butyl)-,trans-),IP-751 ((3R,4R)-(delta 6)-THC-DMH-11-oic acid), PF-5901(benzenemethanol, alpha-pentyl-3-(2-quinolinylmethoxy)-), LY-293111(benzoic acid,24343-((5-ethyl-4′-fluoro-2-hydroxy(1,1′-biphenyl)-4-yl)oxy)propoxy)-2-propylphenoxy)-),RG-5901-A (benzenemethanol, alpha-pentyl-3-(2-quinolinylmethoxy)-,hydrochloride), rilopirox (2(1H)-pyridinone,6-((4-(4-chlorophenoxy)phenoxy)methyl)-1-hydroxy-4-methyl-), L-674636(acetic acid,((4-(4-chlorophenyl)-1-(4-(2-quinolinylmethoxy)phenyl)butyl)thio)-AS)),7-((3-(4-methoxy-tetrahydro-2H-pyran-4-yl)phenyl)methoxy)-4-phenylnaphtho(2,3-c)furan-1(3H)-one,MK-886 (1H-indole-2-propanoic acid,1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-(1-methylethyl)-), quiflapon (1H-indole-2-propanoicacid, 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-), quiflapon(1H-Indole-2-propanoic acid,1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-), docebenone(2,5-cyclohexadiene-1,4-dione,2-(12-hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-), zileuton (urea,N-(1-benzo(b)thien-2-ylethyl)-N-hydroxy-), or an analogue or derivativethereof).

Chemokine Receptor Antagonists CCR (1, 3, and 5)

In another embodiment, the pharmacologically active compound is achemokine receptor antagonist which inhibits one or more subtypes of CCR(1, 3, and 5) (e.g., ONO-4128 (1,4,9-triazaspiro(5.5)undecane-2,5-dione,1-butyl-3-(cyclohexylmethyl)-9-((2,3-dihydro-1,4-benzodioxin-6-yl)methyl-),L-381, CT-112 (L-arginine,L-threonyl-L-threonyl-L-seryl-L-glutaminyl-L-valyl-L-arginyl-L-prolyl-),AS-900004, SCH—C, ZK-811752, PD-172084, UK-427857, SB-380732, vMIP II,SB-265610, DPC-168, TAK-779(N,N-dimethyl-N-(4-(2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-ylcarboxamido)benzyl)tetrahydro-2H-pyran-4-aminiumchloride), TAK-220, KRH-1120), GSK766994, SSR-150106, or an analogue orderivative thereof). Other examples of chemokine receptor antagonistsinclude a-Immunokine-NNS03, BX-471, CCX-282, Sch-350634; Sch-351125;Sch-417690; SCH—C, and analogues and derivatives thereof.

Cell Cycle Inhibitors

In another embodiment, the pharmacologically active compound is a cellcycle inhibitor. Representative examples of such agents include taxanes(e.g., paclitaxel (discussed in more detail below) and docetaxel)(Schiff et al., Nature 277:665-667 (1979); Long and Fairchild, CancerResearch 54:4355-4361 (1994); Ringel and Horwitz, J. Nat'l Cancer Inst.83(4):288-291 (1991); Pazdur et al., Cancer Treat. Rev. 19(40):351-386,1993), etanidazole, nimorazole (B. A. Chabner and D. L. Longo. CancerChemotherapy and Biotherapy—Principles and Practice. Lippincott-RavenPublishers, New York, 1996, p. 554), perfluorochemicals with hyperbaricoxygen, transfusion, erythropoietin, BW 12C, nicotinamide, hydralazine,BSO, WR-2721, IudR, DUdR, etanidazole, WR-2721, BSO, mono-substitutedketo-aldehyde compounds (U.S. Pat. No. 4,066,650 to Egyud),nitroimidazole (U.S. Pat. No. 4,462,992 to Agrawal and Sakaguchi),5-substituted-4-nitroimidazoles (Adams et al., Int. J. Radiat. Biol.Relat. Stud. Phys., Chem. Med. 40(2):153-61 (1981)), SR-2508 (Brown etal., Int. J. Radiat. Oncol., Biol. Phys. 7(6):695-703 (1981)),2H-isoindolediones (U.S. Pat. No. 4,494,547 to Myers), chiral(((2-bromoethyl)-amino)methyl)-nitro-1H-imidazole-1-ethanol (U.S. Pat.Nos. 5,543,527; 4,797,397; and 5,342,959 to Beylin et al.), nitroanilinederivatives (U.S. Pat. No. 5,571,845 to Denny et al.), DNA-affinichypoxia selective cytotoxins (U.S. Pat. No. 5,602,142 toPapadopoulou-Rosenzweig), halogenated DNA ligand (U.S. Pat. No.5,641,764 to Martin), 1,2,4 benzotriazine oxides (U.S. Pat. Nos.5,616,584; 5,624,925; and 5,175,287 to Lee et al.), nitric oxide (U.S.Pat. No. 5,650,442 to Mitchell et al.), 2-nitroimidazole derivatives(U.S. Pat. No. 4,797,397 to Suto et al.; U.S. Pat. No. 5,270,330 to T.Suzuki; U.S. Pat. No. 5,270,330 to T. Suzuki et al.; European Patent No.0 513 351 to Suzuki), fluorine-containing nitroazole derivatives (U.S.Pat. No. 4,927,941 to Kagiya), copper (U.S. Pat. No. 5,100,885 toAbrams), combination modality cancer therapy (U.S. Pat. No. 4,681,091 toPicker et al.), 5-CldC or (d)H₄U or 5-halo-2′-halo-2′-deoxy-cytidine or-uridine derivatives (U.S. Pat. No. 4,894,364 to Greer), platinumcomplexes (U.S. Pat. No. 4,921,963 to Skov; European Publication No. 0287 317 to Skov), fluorine-containing nitroazole (U.S. Pat. No.4,927,941 to Kagiya et al.), benzamide (U.S. Pat. No. 5,032,617 to Lee),autobiotics (U.S. Pat. No. 5,147,652 to Egyud), benzamide andnicotinamide (U.S. Pat. No. 5,215,738 to Lee et al.),acridine-intercalator (U.S. Pat. No. 5,294,715 toPapadopoulou-Rosenzweig), fluorine-containing nitroimidazole (U.S. Pat.No. 5,304,654 to Kagiya et al.), hydroxylated texaphyrins (U.S. Pat. No.5,457,183 to Sessler et al.), hydroxylated compound derivative (JapanesePublication No. 011106775 A to Suzuki et al.; Japanese Publication No.01139596 A to Suzuki et al.; Japanese Publication No. 63170375 A toSakaguchi et al.), fluorine containing 3-nitro-1,2,4-triazole (JapanesePublication No. 02076861 A to Kagitani et al.), 5-thiotretrazolederivative or its salt (Japanese Publication No. 61010511 A to Kano etal.), Nitrothiazole (Japanese Publication No. 61167616 A to Kagitani etal.), imidazole derivatives (Japanese Publication Nos. 6203767 A,62030768 A, and 62030777 A to Inayma et al.), 4-nitro-1,2,3-triazole(Japanese Publication No. 62039525 A to Kagitani et al.),3-nitro-1,2,4-triazole (Japanesee Publication No. 62138427 A to Kagitaniet al.), Carcinostatic action regulator (Japanese Publication No.63099017 A to Amagase), 4,5-dinitroimidazole derivative (JapanesePublication No. 63310873 A to Inayama), nitrotriazole Compound (JapanesePublication No. 07149737 A to Kagitanil), cisplatin, doxorubin,misonidazole, mitomycin, tiripazamine, nitrosourea, mercaptopurine,methotrexate, fluorouracil, bleomycin, vincristine, carboplatin,epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide (I. F.Tannock, Journal of Clinical Oncology 14(12):3156-3174 (1996)),camptothecin (Ewend M. G. et al., Cancer Research 56(22):5217-5223(1996) and paclitaxel (Tishler R. B. et al. International Journal ofRadiation Oncology and Biological Physics 22(3):613-617 (1992)).

A number of the above-mentioned cell cycle inhibitors also have a widevariety of analogues and derivatives, including, but not limited to,cisplatin, cyclophosphamide, misonidazole, tiripazamine, nitrosourea,mercaptopurine, methotrexate, fluorouracil, epirubicin, doxorubicin,vindesine and etoposide. Analogues and derivatives include(CPA)₂Pt(DOLYM) and (DACH)Pt(DOLYM) cisplatin (Choi et al., Arch.Pharmacal Res. 22(2):151-156 (1999),Cis-(PtCl₂(4,7-H-5-methyl-7-oxo)1,2,4(triazolo(1,5-a)pyrimidine)₂)(Navarro et al., J. Med. Chem. 41(3):332-338 (1998),(Pt(cis-1,4-DACH)(trans-Cl₂)(CBDCA))•½MeOH cisplatin (Shamsuddin et al.,Inorg. Chem. 36(25):5969-5971 (1997), 4-pyridoxate diammine hydroxyplatinum (Tokunaga et al., Pharm. Sci. 3(7):353-356 (1997),Pt(II)•••Pt(II) (Pt₂(NHCHN(C(CH₂)(CH₃)))₄) (Navarro et al., Inorg. Chem.35(26):7829-7835 (1996), 254-S cisplatin analogue (Koga et al., Neurol.Res. 18(3):244-247 (1996), o-phenylenediamine ligand bearing cisplatinanalogues (Koeckerbauer & Bednarski, J. Inorg. Biochem. 62(4):281-298(1996), trans,cis-(Pt(OAc)₂I₂(en)) (Kratochwil et al., J. Med. Chem.39(13):2499-2507 (1996), estrogenic 1,2-diarylethylenediamine ligand(with sulfur-containing amino acids and glutathione) bearing cisplatinanalogues (Bednarski, J. Inorg. Biochem. 62(1):75 (1996),cis-1,4-diaminocyclohexane cisplatin analogues (Shamsuddin et al., J.Inorg. Biochem. 61(4):291-301 (1996), 5′ orientational isomer ofcis-(Pt(NH₃)(4-aminoTEMP-O) {d(GpG)}) (Dunham & Lippard, J. Am. Chem.Soc. 117(43): 10702-12 (1995), chelating diamine-bearing cisplatinanalogues (Koeckerbauer & Bednarski, J. Pharm. Sci. 84(7):819-23 (1995),1,2-diarylethyleneamine ligand-bearing cisplatin analogues (Otto et al.,J. Cancer Res. Clin. Oncol. 121(1):31-8 (1995),(ethylenediamine)platinum(II) complexes (Pasini et al., J. Chem. Soc.,Dalton Trans. 4:579-85 (1995), CI-973 cisplatin analogue (Yang et al.,Int. J. Oncol. 5(3):597-602 (1994), cis-diamminedichloroplatinum(II) andits analoguescis-1,1-cyclobutanedicarbosylato(2R)-2-methyl-1,4-butanediam-mineplatinum(II)and cis-diammine(glycolato)platinum (C₁₋aycamp & Zimbrick, J. Inorg.Biochem., 26(4):257-67 (1986); Fan et al., Cancer Res. 48(11):3135-9(1988); Heiger-Bernays et al., Biochemistry 29(36):8461-6, 1990; Kikkawaet al., J. Exp. Clin. Cancer Res. 12(4):233-40 (1993); Murray et al.,Biochemistry 31(47):11812-17 (1992); Takahashi et al., Cancer Chemother.Pharmacol. 33(1):31-5 (1993)),cis-amine-cyclohexylamine-dichloroplatinum(II) (Yoshida et al., Biochem.Pharmacol. 48(4):793-9 (1994)), gem-diphosphonate cisplatin analogues(FR 2683529),(meso-1,2-bis(2,6-dichloro-4-hydroxyplenyl)ethylenediamine)dichloroplatinum(II) (Bednarski et al., J. Med. Chem. 35(23):4479-85(1992)), cisplatin analogues containing a tethered dansyl group (Hartwiget al., J. Am. Chem. Soc. 114(21):8292-3 (1992), platinum(II) polyamines(Siegmann et al., Inorg. Met.-Containing Polym. Mater. (Proc. Am. Chem.Soc. Int. Symp) 335-61 (1990)), cis-(3H)dichloro(ethylenediamine)platinum(II) (Eastman, Anal. Biochem. 197(2):311-15(1991)), trans-diamminedichloroplatinum(II) andcis-(Pt(NH₃)₂(N₃-cytosine)C1) (Bellon & Lippard, Biophys. Chem.35(2-3):179-88 (1990)),3H-cis-1,2-diaminocyclohexanedichloroplatinum(II) and3H-cis-1,2-diaminocyclohexanemalonatoplatinum (II) (Oswald et al., Res.Commun. Chem. Pathol. Pharmacol. 64(1):41-58 (1989)),diaminocarboxylatoplatinum (EPA 296321),trans-(D,1)-1,2-diaminocyclohexane carrier ligand-bearing platinumanalogues (Wyrick & Chaney, J. Labelled Compd. Radiopharm. 25(4):349-57,1988)), aminoalkylaminoanthraquinone-derived cisplatin analogues (Kitovet al., Eur. J. Med. Chem. 23(4):381-3 (1988)), spiroplatin,carboplatin, iproplatin and JM40 platinum analogues (Schroyen et al.,Eur. J. Cancer Clin. Oncol. 24(8):1309-12 (1988)), bidentate tertiarydiamine-containing cisplatinum derivatives (Orbell et al., Inorg. Chim.Acta 152(2):125-34 (1988)), platinum(II), platinum(IV) (Liu & Wang,Shandong Yike Daxue Xuebao 24(1):35-41 (1986)),cis-diammine(1,1-cyclobutanedicarboxylato-)platinum(II) (carboplatin,JM8) and ethylenediammine-malonatoplatinum(II) (JM40) (Begg et al.,Radiother. Oncol. 9(2):157-65 (1987)), JM8 and JM9 cisplatin analogues(Harstrick et al., Int. J. Androl. 10(1); 139-45 (1987)),(NPr4)₂((PtCL4).cis-(PtC12-(NH₂Me)₂)) (Brammer et al., J. Chem. Soc.,Chem. Commun. 6:443-5 (1987)), aliphatic tricarboxylic acid platinumcomplexes (EPA 185225), cis-dichloro(amino acid)(tert-butylamine)platinum(II) complexes (Pasini & Bersanetti, Inorg.Chim. Acta 107(4):259-67 (1985)); 4-hydroperoxycylcophosphamide (Ballardet al., Cancer Chemother. Pharmacol. 26(6):397-402 (1990)),acyclouridine cyclophosphamide derivatives (Zakerinia et al., Helv.Chim. Acta 73(4):912-15 (1990)), 1,3,2-dioxa- and -oxazaphosphorinanecyclophosphamide analogues (Yang et al., Tetrahedron 44(20):6305-14(1988)), CS-substituted cyclophosphamide analogues (Spada, University ofRhode Island Dissertation, 1987), tetrahydrooxazine cyclophosphamideanalogues (Valente, University of Rochester Dissertation, 1988), phenylketone cyclophosphamide analogues (Hales et al., Teratology 39(1):31-7(1989)), phenylketophosphamide cyclophosphamide analogues (Ludeman etal., J. Med. Chem. 29(5):716-27 (1986)), ASTA Z-7557 cyclophosphamideanalogues (Evans et al., Int. J. Cancer 34(6):883-90 (1984)),3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (Tsui etal., J. Med. Chem. 25(9):1106-10 (1982)),2-oxobis(2-β-chloroethylamino)-4-,6-dimethyl-1,3,2-oxazaphosphorinanecyclophosphamide (Carpenter et al., Phosphorus Sulfur 12(3):287-93(1982)), 5-fluoro- and 5-chlorocyclophosphamide (Foster et al., J. Med.Chem. 24(12):1399-403 (1981)), cis- and trans-4-phenylcyclophosphamide(Boyd et al., J. Med. Chem. 23(4):372-5 (1980)),5-bromocyclophosphamide, 3,5-dehydrocyclophosphamide (Ludeman et al., J.Med. Chem. 22(2):151-8 (1979)), 4-ethoxycarbonyl cyclophosphamideanalogues (Foster, J. Pharm. Sci. 67(5):709-10 (1978)),arylaminotetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide cyclophosphamideanalogues (Hamacher, Arch. Pharm. (Weinheim, Ger.) 310(5):J, 428-34(1977)), NSC-26271 cyclophosphamide analogues (Montgomery & Struck,Cancer Treat. Rep. 60(4):J381-93 (1976)), benzo annulatedcyclophosphamide analogues (Ludeman & Zon, J. Med. Chem. 18(12):J1251-3(1975)), 6-trifluoromethylcyclophosphamide (Farmer & Cox, J. Med. Chem.18(11):J1106-10 (1975)), 4-methylcyclophosphamide and6-methycyclophosphamide analogues (Cox et al., Biochem. Pharmacol.24(5):J599-606 (1975)); FCE 23762 doxorubicin derivative (Quaglia etal., J. Liq. Chromatogr. 17(18):3911-3923 (1994)), annamycin (Zou etal., J. Pharm. Sci. 82(11):1151-1154 (1993)), ruboxyl (Rapoport et al.,J. Controlled Release 58(2):153-162 (1999)), anthracycline disaccharidedoxorubicin analogue (Pratesi et al., Clin. Cancer Res. 4(11):2833-2839(1998)), N-(trifluoroacetyl)doxorubicin and4′-O-acetyl-N-(trifluoroacetyl)doxorubicin (Berube & Lepage, Synth.Commun. 28(6):1109-1116 (1998)), 2-pyrrolinodoxorubicin (Nagy et al.,Proc. Nat'l Acad. Sci. U.S.A. 95(4):1794-1799 (1998)), disaccharidedoxorubicin analogues (Arcamone et al., J. Nat'l Cancer Inst.89(16):1217-1223 (1997)),4-demethoxy-7-O-(2,6-dideoxy-4-O-(2,3,6-trideoxy-3-amino-α-L-lyxo-hexopyranosyl)-α-L-lyxo-hexopyranosyl)adriamicinonedoxorubicin disaccharide analogue (Monteagudo et al., Carbohydr. Res.300(1):11-16 (1997)), 2-pyrrolinodoxorubicin (Nagy et al., Proc. Nat'lAcad. Sci. U.S.A. 94(2):652-656 (1997)), morpholinyl doxorubicinanalogues (Duran et al., Cancer Chemother. Pharmacol. 38(3):210-216(1996)), enaminomalonyl-β-alanine doxorubicin derivatives (Seitz et al.,Tetrahedron Lett. 36(9):1413-16 (1995)), cephalosporin doxorubicinderivatives (Vrudhula et al., J. Med. Chem. 38(8):1380-5 (1995)),hydroxyrubicin (Solary et al., Int. J. Cancer 58(1):85-94, 1994),methoxymorpholino doxorubicin derivative (Kuhl et al., Cancer Chemother.Pharmacol. 33(1):10-16 (1993)), (6-maleimidocaproyl)hydrazonedoxorubicin derivative (Willner et al., Bioconjugate Chem. 4(6):521-7(1993)), N-(5,5-diacetoxypent-1-yl) doxorubicin (Chemf & Farquhar, J.Med. Chem. 35(17):3208-14 (1992)), FCE 23762 methoxymorpholinyldoxorubicin derivative (Ripamonti et al., Br. J. Cancer 65(5):703-7(1992)), N-hydroxysuccinimide ester doxorubicin derivatives (Demant etal., Biochim. Biophys. Acta 1118(1):83-90 (1991)), polydeoxynucleotidedoxorubicin derivatives (Ruggiero et al., Biochim. Biophys. Acta1129(3):294-302 (1991)), morpholinyl doxorubicin derivatives (EPA434960), mitoxantrone doxorubicin analogue (Krapcho et al., J. Med.Chem. 34(8):2373-80 (1991)), AD198 doxorubicin analogue (Traganos etal., Cancer Res. 51(14):3682-9 (1991)),4-demethoxy-3′-N-trifluoroacetyldoxorubicin (Horton et al., Drug Des.Delivery 6(2):123-9 (1990)), 4′-epidoxorubicin (Drzewoski et al., Pol.J. Pharmacol. Pharm. 40(2):159-65 (1988); Weenen et al., Eur. J. CancerClin. Oncol. 20(7):919-26 (1984)), alkylating cyanomorpholinodoxorubicin derivative (Scudder et al., J. Nat'l Cancer Inst.80(16):1294-8 (1988)), deoxydihydroiodooxorubicin (EPA 275966),adriblastin (Kalishevskaya et al., Vestn. Mosk. Univ. (Biol. 1) 16:21-7(1988)), 4′-deoxydoxorubicin (Schoelzel et al., Leuk. Res. 10(12):1455-9(1986)), 4-demethyoxy-4′-o-methyldoxorubicin (Giuliani et al., Proc.Int. Congr. Chemother. 16:285-70-285-77 (1983)),3′-deamino-3′-hydroxydoxorubicin (Horton et al., J. Antibiot.37(8):853-8 (1984)), 4-demethyoxy doxorubicin analogues (Barbieri etal., Drugs Exp. Clin. Res. 10(2):85-90 (1984)), N-L-leucyl doxorubicinderivatives (Trouet et al., Anthracyclines (Proc. Int. Symp. TumorPharmacother.) 179-81 (1983)), 3′-deamino-3′-(4-methoxy-1-piperidinyl)doxorubicin derivatives (U.S. Pat. No. 4,314,054),3′-deamino-3′-(4-mortholinyl) doxorubicin derivatives (U.S. Pat. No.4,301,277), 4′-deoxydoxorubicin and 4′-o-methyldoxorubicin (Giuliani etal., Int. J. Cancer 27(1):5-13 (1981)), aglycone doxorubicin derivatives(Chan & Watson, J. Pharm. Sci. 67(12):1748-52 (1978)), SM 5887 (PharmaJapan 1468:20, (1995)), MX-2 (Pharma Japan 1420:19 (1994)),4′-deoxy-13(S)-dihydro-4′-iododoxorubicin (EP 275966), morpholinyldoxorubicin derivatives (EPA 434960),3′-deamino-3′-(4-methoxy-1-piperidinyl) doxorubicin derivatives (U.S.Pat. No. 4,314,054), doxorubicin-14-valerate, morpholinodoxorubicin(U.S. Pat. No. 5,004,606), 3′-deamino-3′-(3″-cyano-4″-morpholinyldoxorubicin; 3′-deamino-3′-(3″-cyano-4″-morpholinyl)-β-dihydroxorubicin;(3′-deamino-3′-(3″-cyano-4″-morpholinyl) daunorubicin;3′-deamino-3′-(3″-cyano-4″-morpholinyl)-3-dihydrodaunorubicin; and3′-deamino-3′-(4″-morpholinyl-5-iminodoxorubicin and derivatives (U.S.Pat. No. 4,585,859), 3′-deamino-3′-(4-methoxy-1-piperidinyl) doxorubicinderivatives (U.S. Pat. No. 4,314,054) and 3-deamino-3-(4-morpholinyl)doxorubicin derivatives (U.S. Pat. No. 4,301,277);4,5-dimethylmisonidazole (Born et al., Biochem. Pharmacol. 43(6):1337-44(1992)), azo and azoxy misonidazole derivatives (Gattavecchia & ToneIli,Int. J. Radiat. Biol. Relat. Stud. Phys., Chem. Med. 45(5):469-77(1984)); RB90740 (Wardman et al., Br. J. Cancer, 74 Suppi. 27:S70-S74(1996)); 6-bromo and 6-chloro-2,3-dihydro-1,4-benzothiazines nitrosoureaderivatives (Rai et al., Heterocyci. Commun. 2(6):587-592 (1996)),diamino acid nitrosourea derivatives (Dulude et al., Bioorg. Med. Chem.Lett. 4(22):2697-700 (1994); Dulude et al., Bioorg. Med. Chem.3(2):151-60 (995), amino acid nitrosourea derivatives (Zheleva et al.,Pharmazie 50(1):25-6 (1995)),3′,4′-didemethoxy-3′,4′-dioxo-4-deoxypodophyllotoxin nitrosoureaderivatives (Miyahara et al., Heterocycles 39(1):361-9 (1994)), ACNU(Matsunaga et al., Immunopharmacology 23(3):199-204 (1992)), tertiaryphosphine oxide nitrosourea derivatives (Guguva et al., Pharmazie46(8):603 (1991)), sulfamerizine and sulfamethizole nitrosoureaderivatives (Chiang et al., Zhonghua Yaozue Zazhi 43(5):401-6 (1991)),thymidine nitrosourea analogues (Zhang et al., Cancer Commun.3(4):119-26, 1991), 1,3-bis(2-chloroethyl)-1-nitrosourea (August et al.,Cancer Res. 51(6):1586-90 (1991)),2,2,6,6-tetramethyl-1-oxopiperidiunium nitrosourea derivatives (U.S.S.R.1261253), 2- and 4-deoxy sugar nitrosourea derivatives (U.S. Pat. No.4,902,791), nitroxyl nitrosourea derivatives (U.S.S.R. 1336489),fotemustine (Boutin et al., Eur. J. Cancer Clin. Oncol. 25(9):1311-16(1989)), pyrimidine (II) nitrosourea derivatives (Wei et al., Chung-huaYao Hsuch Tsa Chih 41(1):19-26 (1989)), CGP 6809 (Schieweck et al.,Cancer Chemother. Pharmacol. 23(6):341-7 (1989)), B-3839 (Prajda et al.,In Vivo 2(2):151-4 (1988)), 5-halogenocytosine nitrosourea derivatives(Chiang & Tseng, T'ai-wan Yao Hsuch Tsa Chih 38(1):37-43 (1986)),1-(2-chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea (Fujimoto &Ogawa, J. Pharmacobio-Dyn. 10(7):341-5 (1987)), sulfur-containingnitrosoureas (Tang et al., Yaoxue Xuebao 21(7):502-9, 1986), sucrose,6-((((2-chloroethyl)nitrosoamino-)carbonyl)amino)-6-deoxysucrose (NS-1C)and 6′-((((2-chloroethyl)nitrosoamino)carbonyl)amino)-6′-deoxysucrose(NS-1D) nitrosourea derivatives (Tanoh et al., Chemotherapy (Tokyo)33(11):969-77 (1985)), CNCC, RFCNU and chlorozotocin (Mena et al.,Chemotherapy (Basel) 32(2):131-7 (1986)), CNUA (Edanami et al.,Chemotherapy (Tokyo) 33(5):455-61 (1985)),1-(2-chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea (Fujimoto &Ogawa, Jpn. J. Cancer Res. (Gann) 76(7):651-6 (1985)), choline-likenitrosoalkylureas (Belyaev et al., Izv. Akad. NAUK SSSR, Ser. Khim.3:553-7 (1985)), sucrose nitrosourea derivatives (JP 84219300), sulfadrug nitrosourea analogues (Chiang et al., Proc. Nat'l Sci. Counc.,Repub. China, Part A 8(1):18-22 (1984)), DONU (Asanuma et al., J. Jpn.Soc. Cancer Ther. 17(8):2035-43 (1982)),N,N′-bis(N-(2-chloroethyl)-N-nitrosocarbamoyl)cystamine (CNCC) (Blazseket al., Toxicol. Appl. Pharmacol. 74(2):250-7 (1984)),dimethylnitrosourea (Krutova et al., Izv. Akad. NAUK SSSR, Ser. Biol.3:439-45 (1984)), GANU (Sava & Giraldi, Cancer Chemother. Pharmacol.10(3):167-9 (1983)), CCNU (Capelli et al., Med., Biol., Environ.11(1):111-16 (1983)), 5-aminomethyl-2′-deoxyuridine nitrosoureaanalogues (Shiau, Shih Ta Hsuch Pao (Taipei) 27:681-9 (1982)), TA-077(Fujimoto & Ogawa, Cancer Chemother. Pharmacol. 9(3):134-9 (1982)),gentianose nitrosourea derivatives (JP 82 80396), CNCC, RFCNU, RPCNU ANDchlorozotocin (CZT) (Marzin et al., INSERM Symp. (Nitrosoureas CancerTreat.) 19:165-74 (1981)), thiocolchicine nitrosourea analogues (George,Shih Ta Hsuch Pao (Taipei) 25:355-62 (1980)), 2-chloroethyl-nitrosourea(Zeller & Eisenbrand, Oncology 38(1):39-42 (1981)), ACNU,(1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoureahydrochloride) (Shibuya et al., Gan To Kagaku Ryoho 7(8):1393-401(1980)), N-deacetylmethyl thiocolchicine nitrosourea analogues (Lin etal., J. Med. Chem. 23(12):1440-2 (1980)), pyridine and piperidinenitrosourea derivatives (Crider et al., J. Med. Chem. 23(8):848-51(1980)), methyl-CCNU (Zimber & Perk, Refu. Vet. 35(1):28 (1978)),phensuzimide nitrosourea derivatives (Crider et al., J. Med. Chem.23(3):324-6 (1980)), ergoline nitrosourea derivatives (Crider et al., J.Med. Chem. 22(1):32-5 (1979)), glucopyranose nitrosourea derivatives (JP78 95917), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (Farmer et al.,J. Med. Chem. 21(6):514-20 (1978)),4-(3-(2-chloroethyl)-3-nitrosoureid-o)-cis-cyclohexanecarboxylic acid(Drewinko et al., Cancer Treat. Rep. 61(8):J1513-18 (1977)), RPCNU (ICIG1163) (Larnicol et al., Biomedicine 26(3):J176-81 (1977)), IOB-252(Sorodoc et al., Rev. Roum. Med., Virol. 28(1):J 55-61 (1977)),1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (Siebert & Eisenbrand,Mutat. Res. 42(1):J45-50 (1977)),1-tetrahydroxycyclopentyl-3-nitroso-3-(2-chloroethyl)-urea (U.S. Pat.No. 4,039,578),d-1-1-(β-chloroethyl)-3-(2-oxo-3-hexahydroazepinyl)-1-nitrosourea (U.S.Pat. No. 3,859,277) and gentianose nitrosourea derivatives (JP57080396); 6-S-aminoacyloxymethyl mercaptopurine derivatives (Harada etal., Chem. Pharm. Bull. 43(10):793-6 (1995)), 6-mercaptopurine (6-MP)(Kashida et al., Biol. Pharm. Bull. 18(11):1492-7 (1995)),7,8-polymethyleneimidazo-1,3,2-diazaphosphorines (Nilov et al.,Mendeleev Commun. 2:67 (1995)), azathioprine (Chifotides et al., J.Inorg. Biochem. 56(4):249-64 (1994)), methyl-D-glucopyranosidemercaptopurine derivatives (Da Silva et al., Eur. J. Med. Chem.29(2):149-52 (1994)) and s-alkynyl mercaptopurine derivatives (Ratsinoet al., Khim.-Farm. Zh. 15(8):65-7 (1981)); indoline ring and a modifiedornithine or glutamic acid-bearing methotrexate derivatives (Matsuoka etal., Chem. Pharm. Bull. 45(7):1146-1150 (1997)), alkyl-substitutedbenzene ring C bearing methotrexate derivatives (Matsuoka et al., Chem.Pharm. Bull. 44(12):2287-2293 (1996)), benzoxazine or benzothiazinemoiety-bearing methotrexate derivatives (Matsuoka et al., J. Med. Chem.40(1):105-111 (1997)), 10-deazaminopterin analogues (DeGraw et al., J.Med. Chem. 40(3):370-376 (1997)), 5-deazaminopterin and5,10-dideazaminopterin methotrexate analogues (Piper et al., J. Med.Chem. 40(3):377-384 (1997)), indoline moiety-bearing methotrexatederivatives (Matsuoka et al., Chem. Pharm. Bull. 44(7):1332-1337(1996)), lipophilic amide methotrexate derivatives (Pignatello et al.,World Meet. Pharm., Biopharm. Pharm. Technol., 563-4, 1995),L-threo-(2S,4S)-4-fluoroglutamic acid and DL-3,3-difluoroglutamicacid-containing methotrexate analogues (Hart et al., J. Med. Chem.39(1):56-65 (1996)), methotrexate tetrahydroquinazoline analogue(Gangjee, et al., J. Heterocycl. Chem. 32(1):243-8 (1995)),N-(α-aminoacyl) methotrexate derivatives (Cheung et al., Pteridines3(1-2):101-2 (1992)), biotin methotrexate derivatives (Fan et al.,Pteridines 3(1-2):131-2 (1992)), D-glutamic acid or D-erythrou,threo-4-fluoroglutamic acid methotrexate analogues (McGuire et al.,Biochem. Pharmacol. 42(12):2400-3, 1991), β,γ-μethano methotrexateanalogues (Rosowsky et al., Pteridines 2(3):133-9, 1991),10-deazaminopterin (10-EDAM) analogue (Braakhuis et al., Chem. Biol.Pteridines, Proc. Int. Symp. Pteridines Folic Acid Deriv. 1027-30(1989)), γ-tetrazole methotrexate analogue (Kalman et al., Chem. Biol.Pteridines, Proc. Int. Symp. Pteridines Folic Acid Deriv. 1154-7(1989)), N-(L-α-aminoacyl) methotrexate derivatives (Cheung et al.,Heterocycles 28(2):751-8 (1989)), meta and ortho isomers of aminopterin(Rosowsky et al., J. Med. Chem. 32(12):2582 (1989)),hydroxymethylmethotrexate (DE 267495), γ-fluoromethotrexate (McGuire etal., Cancer Res. 49(16):4517-25 (1989)), polyglutamyl methotrexatederivatives (Kumar et al., Cancer Res. 46(10):5020-3 (1986)),gem-diphosphonate methotrexate analogues (PCT Publication No. WO88/06158), α- and γ-substituted methotrexate analogues (Tsushima et al.,Tetrahedron 44(17):5375-87 (1988)), 5-methyl-5-deaza methotrexateanalogues (U.S. Pat. No. 4,725,687),Nδ-acyl-Na-(4-amino-4-deoxypteroyl)-L-ornithine derivatives (Rosowsky etal., J. Med. Chem. 31(7):1332-7 (1988)), 8-deaza methotrexate analogues(Kuehl et al., Cancer Res. 48(6):1481-8 (1988)), acivicin methotrexateanalogue (Rosowsky et al., J. Med. Chem. 30(8):1463-9 (1987)), polymericplatinol methotrexate derivative (Carraher et al., Polym. Sci. Technol.(Plenum) (Adv. Biomed. Polym.) 35:311-24 (1987)),methotrexate-γ-dimyristoylphophatidylethanolamine (Kinsky et al.,Biochim. Biophys. Acta 917(2):211-18 (1987)), methotrexate polyglutamateanalogues (Rosowsky et al., Chem. Biol. Pteridines, Pteridines FolidAcid Deriv., Proc. Int. Symp. Pteridines Folid Acid Deriv.: Chem. Biol.Clin. Aspects 985-8 1986)), poly-γ-glutamyl methotrexate derivatives(Kisliuk et al., Chem. Biol. Pteridines, Pteridines Folid Acid Deriv.,Proc. Int. Symp. Pteridines Folid Acid Deriv.: Chem., Biol. Clin.Aspects 989-92 (1986)), deoxyuridylate methotrexate derivatives (Webberet al., Chem. Biol. Pteridines, Pteridines Folid Acid Deriv., Proc. Int.Symp. Pteridines Folid Acid Deriv.: Chem. Biol. Clin. Aspects 659-62(1986)), iodoacetyl lysine methotrexate analogue (Delcamp et al., Chem.Biol. Pteridines, Pteridines Folid Acid Deriv., Proc. Int. Symp.Pteridines Folid Acid Deriv.: Chem. Biol. Clin. Aspects 807-9 (1986)),2, omega.-diaminoalkanoid acid-containing methotrexate analogues(McGuire et al., Biochem. Pharmacol. 35(15):2607-13 (1986)),polyglutamate methotrexate derivatives (Kamen & Winick, Methods Enzymol.122:339-46 (1986)), 5-methyl-5-deaza analogues (Piper et al., J. Med.Chem. (Vitam. Coenzymes, Pt. G) 29(6):1080-7 (1986)), quinazolinemethotrexate analogue (Mastropaolo et al., J. Med. Chem. 29(1):155-8(1986)), pyrazine methotrexate analogue (Lever & Vestal, J. Heterocyci.Chem. 22(1):5-6 (1985)), cysteic acid and homocysteic acid methotrexateanalogues (U.S. Pat. No. 4,490,529), γ-tert-butyl methotrexate esters(Rosowsky et al., J. Med. Chem. 28(5):660-7 (1985)), fluorinatedmethotrexate analogues (Tsushima et al., Heterocycles 23(1):45-9(1985)), folate methotrexate analogue (Trombe, J. Bacteriol.160(3):849-53 (1984)), phosphonoglutamic acid analogues (Sturtz &Guillamot, Eur. J. Med. Chem.—Chim. Ther. 19(3):267-73 (1984)), poly(L-lysine) methotrexate conjugates (Rosowsky et al., J. Med. Chem.27(7):888-93 (1984)), dilysine and trilysine methotrexate derivates(Forsch & Rosowsky, J. Org. Chem. 49(7):1305-9 (1984)),7-hydroxymethotrexate (Fabre et al., Cancer Res. 43(10):4648-52 (1983)),poly-γ-glutamyl methotrexate analogues (Piper & Montgomery, Adv. Exp.Med. Biol. (Folyl Antifolyl Polyglutamates) 163):95-100 (1983)),3′,5′-dichloromethotrexate (Rosowsky & Yu, J. Med. Chem. 26(10):1448-52(1983)), diazoketone and chloromethylketone methotrexate analogues(Gangjee et al., J. Pharm. Sci. 71(6):717-19 (1982)),10-propargylaminopterin and alkyl methotrexate homologs (Piper et al.,J. Med. Chem. 25(7):877-80 (1982)), lectin derivatives of methotrexate(Lin et al., JNCI 66(3):523-8, 1981), polyglutamate methotrexatederivatives (Galivan, Mol. Pharmacol. 17(1):105-10 (1980)), halogentatedmethotrexate derivatives (Fox, JNCI 58(4):J955-8 (1977)),8-alkyl-7,8-dihydro analogues (Chaykovsky et al., J. Med. Chem.20(10):J1323-7 (1977)), 7-methyl methotrexate derivatives anddichloromethotrexate (Rosowsky & Chen, J. Med. Chem. 17(12):J1308-11(1974)), lipophilic methotrexate derivatives and3′,5′-dichloromethotrexate (Rosowsky, J. Med. Chem. 16(10):J1190-3(1973), deaza amethopterin analogues (Montgomery et al., Ann. N.Y. Acad.Sci. 186:J227-34 (1971), MX068 (Pharma Japan, 1658:18 (1999)) andcysteic acid and homocysteic acid methotrexate analogues (EPA 0142220);N3-alkylated analogues of 5-fluorouracil (Kozai et al., J. Chem. Soc.,Perkin Trans. 1(19):3145-3146 (1998), 5-fluorouracil derivatives with1,4-oxaheteroepane moieties (Gomez et al., Tetrahedron54(43):13295-13312 (1998), 5-fluorouracil and nucleoside analogues (Li,Anticancer Res. 17(1A):21-27 (1997), cis- andtrans-5-fluoro-5,6-dihydro-6-alkoxyuracil (Van der Wilt et al., Br. J.Cancer 68(4):702-7 (1993), cyclopentane 5-fluorouracil analogues(Hronowski & Szarek, Can. J. Chem. 70(4):1162-9 (1992),A-OT-fluorouracil (Zhang et al., Zongguo Yiyao Gongye Zazhi20(11):513-15 (1989), N4-trimethoxybenzoyl-5′-deoxy-5-fluorocytidine and5′-deoxy-5-fluorouridine (Miwa et al., Chem. Pharm. Bull. 38(4):998-1003(1990), 1-hexylcarbamoyl-5-fluorouracil (Hoshi et al., J.Pharmacobio-Dun. 3(9):478-81 (1980); Maehara et al., Chemotherapy(Basel) 34(6):484-9 (1988)), B-3839 (Prajda et al., In Vivo 2(2):151-4(1988)), uracil-1-(2-tetrahydrofuryl)-5-fluorouracil (Anai et al.,Oncology 45(3):144-7 (1988)),1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-fluorouracil (Suzuko etal., Mol. Pharmacol. 31(3):301-6 (1987)), doxifluridine (Matuura et al.,Oyo Yakuri 29(5):803-31 (1985)), 5′-deoxy-5-fluorouridine (Bollag &Hartmann, Eur. J. Cancer 16(4):427-32 (1980)),1-acetyl-3-O-toluoyl-5-fluorouracil (Okada, Hiroshima J. Med. Sci.28(1):49-66 (1979)), 5-fluorouracil-m-formylbenzene-sulfonate (JP55059173), N′-(2-furanidyl)-5-fluorouracil (JP 53149985) and1-(2-tetrahydrofuryl)-5-fluorouracil (JP 52089680); 4′-epidoxorubicin(Lanius, Adv. Chemother. Gastrointest. Cancer, (Int. Symp.), 159-67(1984)); N-substituted deacetylvinblastine amide (vindesine) sulfates(Conrad et al., J. Med. Chem. 22(4):391-400 (1979)); and Cu(II)—VP-16(etoposide) complex (Tawa et al., Bioorg. Med. Chem. 6(7):1003-1008(1998)), pyrrolecarboxamidino-bearing etoposide analogues (Ji et al.,Bioorg. Med. Chem. Lett. 7(5):607-612 (1997)), 4β-amino etoposideanalogues (Hu, University of North Carolina Dissertation, 1992),γ-lactone ring-modified arylamino etoposide analogues (Zhou et al., J.Med. Chem. 37(2):287-92 (1994)), N-glucosyl etoposide analogue (Alleviet al., Tetrahedron Lett. 34(45):7313-16 (1993)), etoposide A-ringanalogues (Kadow et al., Bioorg. Med. Chem. Lett. 2(1):17-22 (1992)),4′-deshydroxy-4′-methyl etoposide (Saulnier et al., Bioorg. Med. Chem.Lett. 2(10):1213-18 (1992)), pendulum ring etoposide analogues (Sinha etal., Eur. J. Cancer 26(5):590-3 (1990) and E-ring desoxy etoposideanalogues (Saulnier et al., J. Med. Chem. 32(7):1418-20 (1989)).

Within one preferred embodiment of the invention, the cell cycleinhibitor is paclitaxel, a compound which disrupts mitosis (M-phase) bybinding to tubulin to form abnormal mitotic spindles or an analogue orderivative thereof. Briefly, paclitaxel is a highly derivatizedditerpenoid (Wani et al., J. Am. Chem. Soc. 93:2325 (1971)) which hasbeen obtained from the harvested and dried bark of Taxus brevifolia(Pacific Yew) and Taxomyces Andreanae and Endophytic Fungus of thePacific Yew (Stierle et al., Science 60:214-216 (1993)). “Paclitaxel”(which should be understood herein to include formulations, prodrugs,analogues and derivatives such as, for example, TAXOL® (Bristol MyersSquibb, New York, N.Y., TAXOTERE® (Aventis Pharmaceuticals, France),docetaxel, 10-desacetyl analogues of paclitaxel and3N-desbenzoyl-3′N-t-butoxy carbonyl analogues of paclitaxel) may bereadily prepared utilizing techniques known to those skilled in the art(see, e.g., Schiff et al., Nature 277:665-667 (1979); Long andFairchild, Cancer Research 54:4355-4361 (1994); Ringel and Horwitz, J.Nat'l Cancer Inst. 83(4):288-291 (1991); Pazdur et al., Cancer Treat.Rev. 19(4):351-386 (1993); WO 94/07882; WO 94/07881; WO 94/07880; WO94/07876; WO 93/23555; WO 93/10076; WO 94/00156; WO 93/24476; EP 590267;WO 94/20089; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137;5,202,448; 5,200,534; 5,229,529; 5,254,580; 5,412,092; 5,395,850;5,380,751; 5,350,866; 4,857,653; 5,272,171; 5,411,984; 5,248,796;5,248,796; 5,422,364; 5,300,638; 5,294,637; 5,362,831; 5,440,056;4,814,470; 5,278,324; 5,352,805; 5,411,984; 5,059,699; 4,942,184;Tetrahedron Letters 35(52):9709-9712 (1994); J. Med. Chem. 35:4230-4237(1992); J. Med. Chem. 34:992-998 (1991); J. Natural Prod.57(10):1404-1410 (1994); J. Natural Prod. 57(11):1580-1583 (1994); J.Am. Chem. Soc. 110:6558-6560 (1988)), or obtained from a variety ofcommercial sources, including for example, Sigma Chemical Co., St.Louis, Mo. (T7402—from Taxus brevifolia).

Representative examples of paclitaxel derivatives or analogues include7-deoxy-docetaxol, 7,8-cyclopropataxanes, N-substituted 2-azetidones,6,7-epoxy paclitaxels, 6,7-modified paclitaxels, 10-desacetoxytaxol,10-deacetyltaxol (from 10-deacetylbaccatin III), phosphonooxy andcarbonate derivatives of taxol, taxol 2′,7-di(sodium1,2-benzenedicarboxylate,10-desacetoxy-11,12-dihydrotaxol-10,12(18)-diene derivatives,10-desacetoxytaxol, Protaxol (2′- and/or 7-O-ester derivatives), (2′-and/or 7-O-carbonate derivatives), asymmetric synthesis of taxol sidechain, fluoro taxols, 9-deoxotaxane, (13-acetyl-9-deoxobaccatine III,9-deoxotaxol, 7-deoxy-9-deoxotaxol, 10-desacetoxy-7-deoxy-9-deoxotaxol,Derivatives containing hydrogen or acetyl group and a hydroxy andtert-butoxycarbonylamino, sulfonated 2′-acryloyltaxol and sulfonated2′-O-acyl acid taxol derivatives, succinyltaxol, 2′-γ-aminobutyryltaxolformate, 2′-acetyl taxol, 7-acetyl taxol, 7-glycine carbamate taxol,2′—OH-7-PEG(5000) carbamate taxol, 2′-benzoyl and 2′,7-dibenzoyl taxolderivatives, other prodrugs (2′-acetyltaxol; 2′,7-diacetyltaxol; 2′succinyltaxol; 2′-(beta-alanyl)-taxol); 2′-γ-aminobutyryltaxol formate;ethylene glycol derivatives of 2′-succinyltaxol; 2′-glutaryltaxol;2′-(N,N-dimethylglycyl) taxol; 2′-(2-(N,N-dimethylamino)propionyl)taxol;2′ orthocarboxybenzoyl taxol; 2′ aliphatic carboxylic acid derivativesof taxol, Prodrugs {2′(N,N-diethylaminopropionyl)taxol,2′(N,N-dimethylglycyl)taxol, 7(N,N-dimethylglycyl)taxol,2′,7-di-(N,N-dimethylglycyl)taxol, 7(N,N-diethylaminopropionyl)taxol,2′,7-di(N,N-diethylaminopropionyl)taxol, 2′-(L-glycyl)taxol,7-(L-glycyl)taxol, 2′,7-di(L-glycyl)taxol, 2′-(L-alanyl)taxol,7-(L-alanyl)taxol, 2′,7-di(L-alanyl)taxol, 2′-(L-leucyl)taxol,7-(L-leucyl)taxol, 2′,7-di(L-leucyl)taxol, 2′-(L-isoleucyl)taxol,7-(L-isoleucyl)taxol, 2′,7-di(L-isoleucyl)taxol, 2′-(L-valyl)taxol,7-(L-valyl)taxol, 2′7-di(L-valyl)taxol, 2′-(L-phenylalanyl)taxol,7-(L-phenylalanyl)taxol, 2′,7-di(L-phenylalanyl)taxol,2′-(L-prolyl)taxol, 7-(L-prolyl)taxol, 2′,7-di(L-prolyl)taxol,2′-(L-lysyl)taxol, 7-(L-lysyl)taxol, 2′,7-di(L-lysyl)taxol,2′-(L-glutamyl)taxol, 7-(L-glutamyl)taxol, 2′,7-di(L-glutamyl)taxol,2′-(L-arginyl)taxol, 7-(L-arginyl)taxol, 2′,7-di(L-arginyl)taxol}, taxolanalogues with modified phenylisoserine side chains, TAXOTERE®,(N-debenzoyl-N-tert-(butoxycaronyl)-10-deacetyltaxol, and taxanes (e.g.,baccatin III, cephalomannine, 10-deacetylbaccatin III, brevifoliol,yunantaxusin and taxusin); and other taxane analogues and derivatives,including 14-beta-hydroxy-10 deacetybaccatin III, debenzoyl-2-acylpaclitaxel derivatives, benzoate paclitaxel derivatives, phosphonooxyand carbonate paclitaxel derivatives, sulfonated 2′-acryloyltaxol;sulfonated 2′-O-acyl acid paclitaxel derivatives, 18-site-substitutedpaclitaxel derivatives, chlorinated paclitaxel analogues, C4 methoxyether paclitaxel derivatives, sulfonamide taxane derivatives, brominatedpaclitaxel analogues, Girard taxane derivatives, nitrophenyl paclitaxel,10-deacetylated substituted paclitaxel derivatives, 14-beta-hydroxy-10deacetylbaccatin III taxane derivatives, C7 taxane derivatives, C10taxane derivatives, 2-debenzoyl-2-acyl taxane derivatives, 2-debenzoyland -2-acyl paclitaxel derivatives, taxane and baccatin III analoguesbearing new C2 and C4 functional groups, n-acyl paclitaxel analogues,10-deacetylbaccatin III and 7-protected-10-deacetylbaccatin IIIderivatives from 10-deacetyl taxol A, 10-deacetyl taxol B, and10-deacetyl taxol, benzoate derivatives of taxol, 2-aroyl-4-acylpaclitaxel analogues, orthro-ester paclitaxel analogues, 2-aroyl-4-acylpaclitaxel analogues and 1-deoxy paclitaxel and 1-deoxy paclitaxelanalogues.

In one aspect, the cell cycle inhibitor is a taxane having the structureof formula (IV):

wherein the circled area may be substituted and the non-circled portionis the taxane core. A side-chain (labeled “A” in the diagram) isdesirably present in order for the compound to have good activity as acell cycle inhibitor. Examples of compounds having this structureinclude paclitaxel (Merck Index entry 7117), docetaxol (TAXOTERE, MerckIndex entry 3458), and3′-desphenyl-3′-(4-ntirophenyl)-N-debenzoyl-N-(t-butoxycarbonyl)-10-deacetyltaxol.

In one aspect, suitable taxanes such as paclitaxel and its analogues andderivatives are disclosed in U.S. Pat. No. 5,440,056 as having thestructure of formula (V):

wherein X may be oxygen (paclitaxel), hydrogen (9-deoxy derivatives),thioacyl, or dihydroxyl precursors; R₁ is selected from paclitaxel orTAXOTERE side chains or alkanoyl having the structure of formula (VI)

wherein R₇ is selected from hydrogen, alkyl, phenyl, alkoxy, amino,phenoxy (substituted or unsubstituted); R₈ is selected from hydrogen,alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, phenyl (substituted orunsubstituted), alpha or beta-naphthyl; and R₉ is selected fromhydrogen, alkanoyl, substituted alkanoyl, and aminoalkanoyl; wheresubstitutions refer to hydroxyl, sulfhydryl, allalkoxyl, carboxyl,halogen, thioalkoxyl, N,N-dimethylamino, alkylamino, dialkylamino,nitro, and —OSO₃H, and/or may refer to groups containing suchsubstitutions; R₂ is selected from hydrogen or oxygen-containing groups,such as hydrogen, hydroxyl, alkoyl, alkanoyloxy, aminoalkanoyloxy, andpeptidyalkanoyloxy; R₃ is selected from hydrogen or oxygen-containinggroups, such as hydrogen, hydroxyl, alkoyl, alkanoyloxy,aminoalkanoyloxy, and peptidyalkanoyloxy, and may further be a silylcontaining group or a sulphur containing group; R₄ is selected fromacyl, alkyl, alkanoyl, aminoalkanoyl, peptidylalkanoyl and aroyl; R₅ isselected from acyl, alkyl, alkanoyl, aminoalkanoyl, peptidylalkanoyl andaroyl; R₆ is selected from hydrogen or oxygen-containing groups, such ashydrogen, hydroxyl alkoyl, alkanoyloxy, aminoalkanoyloxy, andpeptidyalkanoyloxy.

In one aspect, the paclitaxel analogues and derivatives useful as cellcycle inhibitors are disclosed in PCT International Patent ApplicationNo. WO 93/10076. As disclosed in this publication, the analogue orderivative should have a side chain attached to the taxane nucleus atC₁₃, as shown in the structure of formula VII, in order to conferantitumor activity to the taxane.

WO 93/10076 discloses that the taxane nucleus may be substituted at anyposition with the exception of the existing methyl groups. Thesubstitutions may include, for example, hydrogen, alkanoyloxy,alkenoyloxy, aryloyloxy. In addition, oxo groups may be attached tocarbons labeled 2, 4, 9, and/or 10. As well, an oxetane ring may beattached at carbons 4 and 5. As well, an oxirane ring may be attached tothe carbon labeled 4.

In one aspect, the taxane-based cell cycle inhibitor useful in thepresent invention is disclosed in U.S. Pat. No. 5,440,056, whichdiscloses 9-deoxo taxanes. These are compounds lacking an oxo group atthe carbon labeled 9 in the taxane structure shown above (formula VI).The taxane ring may be substituted at the carbons labeled 1, 7, and 10(independently) with H, OH, O—R, or O—CO—R where R is an alkyl or anaminoalkyl. As well, it may be substituted at carbons labeled 2 and 4(independently) with aryol, alkanoyl, aminoalkanoyl, or alkyl groups.The side chain of formula (V) may be substituted at R₇ and R₈(independently) with phenyl rings, substituted phenyl rings, linearalkanes/alkenes, and groups containing H, O, or N. R₉ may be substitutedwith H, or a substituted or unsubstituted alkanoyl group.

Taxanes in general, and paclitaxel is particular, is considered tofunction as a cell cycle inhibitor by acting as an anti-microtubuleagent, and more specifically as a stabilizer. These compounds have beenshown useful in the treatment of proliferative disorders, including:non-small cell (NSC) lung; small cell lung; breast; prostate; cervical;endometrial; head and neck cancers.

In another aspect, the anti-microtuble agent (microtubule inhibitor) isalbendazole (carbamic acid, [5-(propylthio)-1H-benzimidazol-2-yl]-,methyl ester), LY-355703(1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetrone,10-[(3-chloro-4-methoxyphenyl)methyl]-6,6-dimethyl-3-(2-methylpropyl)-16-[(1S)-1-[(2S,3R)-3-phenyloxiranyl]ethyl]-,(3S,10R,13E,16S)—), vindesine (vincaleukoblastine,3-(aminocarbonyl)-O4-deacetyl-3-de(methoxycarbonyl)-), or WAY-174286

In another aspect, the cell cycle inhibitor is a vinca alkaloid. Vincaalkaloids have the following general structure of formulas (VIII) and(IX). They are indole-dihydroindole dimers.

As disclosed in U.S. Pat. Nos. 4,841,045 and 5,030,620, R₁ can be aformyl or methyl group or alternately H. R₁ can also be an alkyl groupor an aldehyde-substituted alkyl (e.g., CH₂CHO). R₂ is typically a CH₃or NH₂ group; however it can be alternately substituted with a loweralkyl ester or the ester linking to the dihydroindole core may besubstituted with C(O)—R where R is NH₂, an amino acid ester or a peptideester. R₃ is typically C(O)CH₃, CH₃, or H. Alternately, a proteinfragment may be linked by a bifunctional group, such as maleoyl aminoacid. R₃ can also be substituted to form an alkyl ester which may befurther substituted. R₄ may be —CH₂— or a single bond. R₅ and R₆ may beH, OH or a lower alkyl, typically —CH₂CH₃. Alternatively R₆ and R₇ maytogether form an oxetane ring. R₇ may alternately be H. Furthersubstitutions include molecules wherein methyl groups are substitutedwith other alkyl groups, and whereby unsaturated rings may bederivatized by the addition of a side group such as an alkane, alkene,alkyne, halogen, ester, amide, or amino group.

Exemplary vinca alkaloids are vinblastine, vincristine, vincristinesulfate, vindesine, and vinorelbine, having the structures of formula(X):

(X)

R₁ R₂ R₃ R₄ R₅ Vinblastine: CH₃ CH₃ C(O)CH₃ OH CH₂ Vincristine: CH₂O CH₃C(O)CH₃ OH CH₂ Vindesine: CH₃ NH₂ H OH CH₂ Vinorelbine: CH₃ CH₃ CH₃ Hsingle bond

Analogues typically require the side group (circled area) in order tohave activity. These compounds are thought to act as cell cycleinhibitors by functioning as anti-microtubule agents, and morespecifically to inhibit polymerization. These compounds have been shownuseful in treating proliferative disorders, including NSC lung; smallcell lung; breast; prostate; brain; head and neck; retinoblastoma;bladder; and penile cancers; and soft tissue sarcoma.

In another aspect, the cell cycle inhibitor is a camptothecin, or ananalog or derivative thereof. Camptothecins have the following generalstructure of formula (XI):

In this structure, X is typically O, but can be other groups, e.g., NHin the case of 21-lactam derivatives. R₁ is typically H or OH, but maybe other groups, e.g., a terminally hydroxylated C₁₋₃ alkane. R₂ istypically H or an amino containing group such as (CH₃)₂NHCH₂, but may beother groups e.g., NO₂, NH₂, halogen (as disclosed in, e.g., U.S. Pat.No. 5,552,156) or a short alkane containing these groups. R₃ istypically H or a short alkyl such as C₂H₅. R₄ is typically H but may beother groups, e.g., a methylenedioxy group with R₁.

Exemplary camptothecin compounds include topotecan, irinotecan (CPT-11),9-aminocamptothecin, 21-lactam-20(S)-camptothecin,10,11-methylenedioxycamptothecin, SN-38, 9-nitrocamptothecin,10-hydroxycamptothecin. Exemplary compounds have the structures offormula (XII):

(XII)

R₁ R₂ R₃ Camptothecin: H H H Topotecan: OH (CH₃)₂NHCH₂ H SN-38: OH HC₂H₅ X: O for most analogs, NH for 21-lactam analogs

Camptothecins have the five rings shown here. The ring labeled E must beintact (the lactone rather than carboxylate form) for maximum activityand minimum toxicity. These compounds are useful to as cell cycleinhibitors, where they can function as topoisomerase I inhibitors and/orDNA cleavage agents. They have been shown useful in the treatment ofproliferative disorders, including, for example, NSC lung; small celllung; and cervical cancers.

In another aspect, the cell cycle inhibitor is a podophyllotoxin, or aderivative or an analogue thereof. Exemplary compounds of this type areetoposide or teniposide, which have the following structures of formula(XIII):

(XIII)

R Etoposide CH₃ Teniposide

These compounds are thought to function as cell cycle inhibitors bybeing topoisomerase II inhibitors and/or by DNA cleaving agents. Theyhave been shown useful as antiproliferative agents in, e.g., small celllung, prostate, and brain cancers, and in retinoblastoma.

Another example of a DNA topoisomerase inhibitor is lurtotecandihydrochloride (11H-1,4-dioxin° [2,3-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinoline-9,12(8H,14H)-dione,8-ethyl-2,3-dihydro-8-hydroxy-15-[(4-methyl-1-piperazinyl)methyl]-,dihydrochloride, (S)—).

In another aspect, the cell cycle inhibitor is an anthracycline.Anthracyclines have the following general structure of formula (XIV),where the R groups may be a variety of organic groups:

According to U.S. Pat. No. 5,594,158, suitable R groups are: R₁ is CH₃or CH₂OH; R₂ is daunosamine or H; R₃ and R₄ are independently one of OH,NO₂, NH₂, F, Cl, Br, I, CN, H or groups derived from these; R₅₋₇ are allH or R₅ and R₆ are H and R₇ and R₈ are alkyl or halogen, or vice versa:R₇ and R₈ are H and R₅ and R₆ are alkyl or halogen.

According to U.S. Pat. No. 5,843,903, R₂ may be a conjugated peptide.According to U.S. Pat. Nos. 4,215,062 and 4,296,105, R₅ may be OH or anether linked alkyl group. R₁ may also be linked to the anthracyclinering by a group other than C(O), such as an alkyl or branched alkylgroup having the C(O) linking moiety at its end, such as—CH₂CH(CH₂—X)C(O)—R₁, wherein X is H or an alkyl group (see, e.g., U.S.Pat. No. 4,215,062). R₂ may alternately be a group linked by thefunctional group ═N—NHC(O)—Y, where Y is a group such as a phenyl orsubstituted phenyl ring. Alternately R₃ may have the following structureof formula (XV):

wherein R₉ is OH either in or out of the plane of the ring, or is asecond sugar moiety such as R₃. R₁₀ may be H or form a secondary aminewith a group such as an aromatic group, saturated or partially saturated5 or 6 membered heterocyclic having at least one ring nitrogen (see U.S.Pat. No. 5,843,903).

Alternately, R₁₀ may be derived from an amino acid, having the structure—C(O)CH(NHR₁₁)(R₁₂), in which R₁₁ is H, or forms a C₃₋₄ memberedalkylene with R₁₂. R₁₂ may be H, alkyl, aminoalkyl, amino, hydroxy,mercapto, phenyl, benzyl or methylthio (see U.S. Pat. No. 4,296,105).

Exemplary anthracyclines are doxorubicin, daunorubicin, idarubicin,epirubicin, pirarubicin, zorubicin, and carubicin. Suitable compoundshave the structures of formula (XVI):

(XVI)

R₁ R₂ R₃ Doxorubicin: OCH₃ CH₂OH OH out of ring plane Epirubicin: OCH₃CH₂OH OH in ring plane (4′ epimer of doxorubicin) Daunorubicin: OCH₃ CH₃OH out of ring plane Idarubicin: H CH₃ OH out of ring plane PirarubicinOCH₃ OH A Zorubicin OCH₃ ═N—NHC(O)C₆H₅ B Carubicin OH CH₃ B A:

B:

Other suitable anthracyclins are anthramycin, mitoxantrone, menogaril,nogalamycin, aclacinomycin A, olivomycin A, chromomycin A₃, andplicamycin having the structures:

These compounds are thought to function as cell cycle inhibitors bybeing topoisomerase inhibitors and/or by DNA cleaving agents. They havebeen shown useful in the treatment of proliferative disorders, includingsmall cell lung; breast; endometrial; head and neck; retinoblastoma;liver; bile duct; islet cell; and bladder cancers; and soft tissuesarcoma.

In another aspect, the cell cycle inhibitor is a platinum compound. Ingeneral, suitable platinum complexes may be of Pt(II) or Pt(IV) and havethis basic structure of formula (XVII):

wherein X and Y are anionic leaving groups such as sulfate, phosphate,carboxylate, and halogen; R₁ and R₂ are alkyl, amine, amino alkyl anymay be further substituted, and are basically inert or bridging groups.For Pt(II) complexes Z₁ and Z₂ are non-existent. For Pt(IV) Z₁ and Z₂may be anionic groups such as halogen, hydroxy, carboxylate, ester,sulfate or phosphate. See, e.g., U.S. Pat. Nos. 4,588,831 and 4,250,189.

Suitable platinum complexes may contain multiple Pt atoms. See, e.g.,U.S. Pat. Nos. 5,409,915 and 5,380,897. For example bisplatinum andtriplatinum complexes of the type:

Exemplary platinum compounds are cisplatin, carboplatin, oxaliplatin,and miboplatin having the structures:

These compounds are thought to function as cell cycle inhibitors bybinding to DNA, i.e., acting as alkylating agents of DNA. Thesecompounds have been shown useful in the treatment of cell proliferativedisorders, including, e.g., NSC lung; small cell lung; breast; cervical;brain; head and neck; esophageal; retinoblastom; liver; bile duct;bladder; penile; and vulvar cancers; and soft tissue sarcoma.

In another aspect, the cell cycle inhibitor is a nitrosourea.Nitrosourease have the following general formula (XVIII), where typicalR groups are shown below.

Other suitable R groups include cyclic alkanes, alkanes, halogensubstituted groups, sugars, aryl and heteroaryl groups, phosphonyl andsulfonyl groups. As disclosed in U.S. Pat. No. 4,367,239, R may suitablybe CH₂—C(X)(Y)(Z), wherein X and Y may be the same or different membersof the following groups: phenyl, cyclyhexyl, or a phenyl or cyclohexylgroup substituted with groups such as halogen, lower alkyl (C₁₋₄),trifluore methyl, cyano, phenyl, cyclohexyl, lower alkyloxy (C₁₀. Z hasthe following structure: -alkylene-N—R₁R₂, where R₁ and R₂ may be thesame or different members of the following group: lower alkyl (C₁₋₄) andbenzyl, or together R₁ and R₂ may form a saturated 5 or 6 memberedheterocyclic such as pyrrolidine, piperidine, morfoline, thiomorfoline,N-lower alkyl piperazine, where the heterocyclic may be optionallysubstituted with lower alkyl groups.

As disclosed in U.S. Pat. No. 6,096,923, R and R′ of formula (XVI) maybe the same or different, where each may be a substituted orunsubstituted hydrocarbon having 1-10 carbons. Substitutions may includehydrocarbyl, halo, ester, amide, carboxylic acid, ether, thioether, andalcohol groups. As disclosed in U.S. Pat. No. 4,472,379, R of formula(XVI) may be an amide bond and a pyranose structure (e.g., methyl2′-(N—(N-(2-chloroethyl)-N-nitroso-carbamoyl)-glycyl)amino-2′-deoxy-α-D-glucopyranoside).As disclosed in U.S. Pat. No. 4,150,146, R of formula (XVI) may be analkyl group of 2 to 6 carbons and may be substituted with an ester,sulfonyl, or hydroxyl group. It may also be substituted with acarboxylic acid or CONH₂ group.

Exemplary nitrosoureas are BCNU (carmustine), methyl-CCNU (semustine),CCNU (lomustine), ranimustine, nimustine, chlorozotocin, fotemustine,and streptozocin, having the following structures

These nitrosourea compounds are thought to function as cell cycleinhibitors by binding to DNA, that is, by functioning as DNA alkylatingagents. These cell cycle inhibitors have been shown useful in treatingcell proliferative disorders such as, for example, islet cell; smallcell lung; melanoma; and brain cancers.

In another aspect, the cell cycle inhibitor is a nitroimidazole, whereexemplary nitroimidazoles are metronidazole, benznidazole, etanidazole,and misonidazole, having the structure of formula (XIX):

(XIX)

R₁ R₂ R₃ Metronidazole OH CH₃ NO₂ Benznidazole C(O)NHCH₂-benzyl NO₂ HEtanidazole CONHCH₂CH₂OH NO₂ H

Suitable nitroimidazole compounds are disclosed in, e.g., U.S. Pat. Nos.4,371,540 and 4,462,992.

In another aspect, the cell cycle inhibitor is a folic acid antagonist,such as methotrexate or derivatives or analogues thereof, includingedatrexate, trimetrexate, raltitrexed, piritrexim, denopterin, tomudex,and pteropterin. Methotrexate analogues have the following generalstructure of formula (XX):

The identity of the R group may be selected from organic groups,particularly those groups set forth in U.S. Pat. Nos. 5,166,149 and5,382,582. For example, R₁ may be N, R₂ may be N or C(CH₃), R₃ and R₃′may H or alkyl, e.g., CH₃, R₄ may be a single bond or NR, where R is Hor alkyl group. R_(5,6,8) may be H, OCH₃, or alternately they can behalogens or hydro groups. R₇ is a side chain of the general structure offormula (XXI):

wherein n=1 for methotrexate, n=3 for pteropterin. The carboxyl groupsin the side chain may be esterified or form a salt such as a Zn²⁺ salt.R₉ and R₁₀ can be NH₂ or may be alkyl substituted.

Exemplary folic acid antagonist compounds have the structures offormulas (XXII) and (XXIII):

(XXII)

R₀ R₁ R₂ R₃ R₄ R₅ R₆ R₇ R₈ Methotrexate NH₂ N N H N(CH₃) H H A (n = 1) HEdatrexate NH₂ N N H N(CH₂CH₃) H H A (n = 1) H Trimetrexate NH₂ N C(CH₃)H NH H OCH₃ OCH₃ OCH₃ Pteropterin NH₂ N N H N(CH₃) H H A (n = 3) HDenopterin OH N N CH₃ N(CH₃) H H A (n = 1) H Piritrexim NH₂ N C(CH₃) Hsingle OCH₃ H H OCH₃ H bond

These compounds are thought to function as cell cycle inhibitors byserving as antimetabolites of folic acid. They have been shown useful inthe treatment of cell proliferative disorders including, for example,soft tissue sarcoma, small cell lung, breast, brain, head and neck,bladder, and penile cancers.

In another aspect, the cell cycle inhibitor is a cytidine analogue, suchas cytarabine or derivatives or analogues thereof, includingenocitabine, FMdC ((E(-2′-deoxy-2′-(fluoromethylene)cytidine),gemcitabine, 5-azacitidine, ancitabine, and 6-azauridine. Exemplarycompounds have the structure of formula (XXIV):

(XXIV)

R₁ R₂ R₃ R₄ Cytarabine H OH H CH Enocitabine C(O)(CH₂)₂₀CH₃ OH H CHGemcitabine H F F CH Azacitidine H H OH N FMdC H CH₂F H CH

These compounds are thought to function as cell cycle inhibitors asacting as antimetabolites of pyrimidine. These compounds have been shownuseful in the treatment of cell proliferative disorders including, forexample, pancreatic, breast, cervical, NSC lung, and bile duct cancers.

In another aspect, the cell cycle inhibitor is a pyrimidine analogue. Inone aspect, the pyrimidine analogues have the general structure offormula (XXV):

wherein positions 2′, 3′, and 5′ on the sugar ring (R₂, R₃ and R₄,respectively) can be H, hydroxyl, phosphoryl (see, e.g., U.S. Pat. No.4,086,417) or ester (see, e.g., U.S. Pat. No. 3,894,000). Esters can beof alkyl, cycloalkyl, aryl, or heterocyclo/aryl types. The 2′ carbon canbe hydroxylated at either R₂ or R₂′, the other group is H. Alternately,the 2′ carbon can be substituted with halogens e.g., fluoro or difluorocytidines such as Gemcytabine. Alternately, the sugar can be substitutedfor another heterocyclic group such as a furyl group or for an alkane,an alkyl ether or an amide linked alkane such as C(O)NH(CH₂)₅CH₃. The 2°amine can be substituted with an aliphatic acyl (R₁) linked with anamide (see, e.g., U.S. Pat. No. 3,991,045) or urethane (see, e.g., U.S.Pat. No. 3,894,000) bond. It can also be further substituted to form aquaternary ammonium salt. R₅ in the pyrimidine ring may be N or CR,where R is H, halogen-containing groups, or alkyl (see, e.g., U.S. Pat.No. 4,086,417). R₆ and R₇ can together can form an oxo group orR₆=—NH—R₁ and R₇═H. R₈ is H or R₇ and R₈ together can form a double bondor R₈ can be X, where X is a structure of formula (XXVI):

Specific pyrimidine analogues are disclosed in U.S. Pat. No. 3,894,000(see, e.g., 2′-O-palmityl-ara-cytidine, 3′-O-benzoyl-ara-cytidine, andmore than 10 other examples); U.S. Pat. No. 3,991,045 (see, e.g.,N4-acyl-1-β-D-arabinofuranosylcytosine, and numerous acyl groupsderivatives as listed therein, such as palmitoyl.

In another aspect, the cell cycle inhibitor is a fluoropyrimidineanalogue, such as 5-fluorouracil, or an analogue or derivative thereof,including carmofur, doxifluridine, emitefur, tegafur, and floxuridine.Exemplary compounds have the structures of formulas (XXVII):

(XXVII)

R₁ R₂ 5-Fluorouracil H H Carmofur C(O)NH(CH₂)₅CH₃ H Doxifluridine A₁ HFloxuridine A₂ H Emitefur CH₂OCH₂CH₃ B Tegafur C H

Other suitable fluoropyrimidine analogues include 5-FudR(5-fluoro-deoxyuridine), or an analogue or derivative thereof, including5-iododeoxyuridine (5-IudR),5-bromodeoxyuridine (5-BudR), fluorouridinetriphosphate (5-FUTP), and fluorodeoxyuridine monophosphate (5-dFUMP).Exemplary compounds have the structures of formula (XXVIII):

These compounds are thought to function as cell cycle inhibitors byserving as antimetabolites of pyrimidine. These compounds have beenshown useful in the treatment of cell proliferative disorders such asbreast, cervical, non-melanoma skin, head and neck, esophageal, bileduct, pancreatic, islet cell, penile, and vulvar cancers.

In another aspect, the cell cycle inhibitor is a purine analogue. Purineanalogues have the following general structure of formula (XXIX):

wherein X is typically carbon; R₁ is H, halogen, amine or a substitutedphenyl; R₂ is H, a primary, secondary or tertiary amine, a sulfurcontaining group, typically —SH, an alkane, a cyclic alkane, aheterocyclic or a sugar; R₃ is H, a sugar (typically a furanose orpyranose structure), a substituted sugar or a cyclic or heterocyclicalkane or aryl group. See, e.g., U.S. Pat. No. 5,602,140 for compoundsof this type.

In the case of pentostatin, X—R2 is —CH₂CH(OH)—. In this case a secondcarbon atom is inserted in the ring between X and the adjacent nitrogenatom. The X—N double bond becomes a single bond.

U.S. Pat. No. 5,446,139 describes suitable purine analogues of the typeshown in the structure of formula (XXX):

wherein N signifies nitrogen and V, W, X, Z can be either carbon ornitrogen with the following provisos. Ring A may have 0 to 3 nitrogenatoms in its structure. If two nitrogens are present in ring A, one mustbe in the W position. If only one is present, it must not be in the Qposition. V and Q must not be simultaneously nitrogen. Z and Q must notbe simultaneously nitrogen. If Z is nitrogen, R₃ is not present.Furthermore, R₁₋₃ are independently one of H, halogen, C₁₋₇ alkyl, C₁₋₇alkenyl, hydroxyl, mercapto, C₁₋₇ alkylthio, C₁₋₇ alkoxy, C₂₋₇alkenyloxy, aryl oxy, nitro, primary, secondary or tertiary aminecontaining group. R₅₋₈ are H or up to two of the positions may containindependently one of OH, halogen, cyano, azido, substituted amino, R₅and R₇ can together form a double bond. Y is H, a C₁₋₇ alkylcarbonyl, ora mono- di or tri phosphate.

Exemplary suitable purine analogues include 6-mercaptopurine,thiguanosine, thiamiprine, cladribine, fludaribine, tubercidin,puromycin, pentoxyfilline; where these compounds may optionally bephosphorylated. Exemplary compounds have the structures of formulas(XXXI) and (XXXII):

(XXXI)

R₁ R₂ R₃ 6-Mercaptopurine H SH H Thioguanosine NH₂ SH B₁ Thiamiprine NH₂A H Cladribine Cl NH₂ B₂ Fludarabine F NH₂ B₃ Puromycin H N(CH₃)₂ B₄Tubercidin H NH₂ B₁

These compounds are thought to function as cell cycle inhibitors byserving as antimetabolites of purine.

In another aspect, the cell cycle inhibitor is a nitrogen mustard. Manysuitable nitrogen mustards are known and are suitably used as a cellcycle inhibitor in the present invention. Suitable nitrogen mustards arealso known as cyclophosphamides.

A preferred nitrogen mustard has the general structure of formula(XXXIV):

wherein A is:

or —CH₃ or other alkane, or chloronated alkane, typically CH₂CH(CH₃)Cl,or a polycyclic group such as B, or a substituted phenyl such as C or aheterocyclic group such as D.

Examples of suitable nitrogen mustards are disclosed in U.S. Pat. No.3,808,297, wherein A is:

wherein R₁₋₂ are H or CH₂CH₂Cl; R₃ is H or oxygen-containing groups suchas hydroperoxy; and R₄ can be alkyl, aryl, heterocyclic. The cyclicmoiety need not be intact. See, e.g., U.S. Pat. Nos. 5,472,956,4,908,356, 4,841,085 that describe the following type of structure offormula (XXXV):

wherein R₁ is H or CH₂CH₂Cl, and R₂₋₆ are various substituent groups.

Exemplary nitrogen mustards include methylchloroethamine, and analoguesor derivatives thereof, including methylchloroethamine oxidehydrohchloride, novembichin, and mannomustine (a halogenated sugar).Exemplary compounds have the following structures:

The nitrogen mustard may be cyclophosphamide, ifosfamide, perfosfamide,or torofosfamide, where these compounds have the structures of formula(XXXVI):

(XXXVI)

R₁ R₂ R₃ Cyclophosphamide H CH₂CH₂Cl H Ifosfamide CH₂CH₂Cl H HPerfosfamide CH₂CH₂Cl H OOH Torofosfamide CH₂CH₂Cl CH₂CH₂Cl H

The nitrogen mustard may be estramustine, or an analogue or derivativethereof, including phenesterine, prednimustine, and estramustine PO₄.Thus, suitable nitrogen mustard type cell cycle inhibitors of thepresent invention have the structures of formulas (XXXVII) and(XXXVIII):

(XXXVII)

R Estramustine OH Phenesterine C(CH₃)(CH₂)₃CH(CH₃)₂

The nitrogen mustard may be chlorambucil, or an analogue or derivativethereof, including melphalan and chlormaphazine. Thus, suitable nitrogenmustard type cell cycle inhibitors of the present invention have thestructures of formula (XXXIX):

(XXXIX)

R₁ R₂ R₃ Chlorambucil CH₂COOH H H Melphalan COOH NH₂ H Chlornaphazine Htogether forms a benzene ring

The nitrogen mustard may be uracil mustard, which has the structure offormula (LX):

The nitrogen mustards are thought to function as cell cycle inhibitorsby serving as alkylating agents for DNA. Nitrogen mustards have beenshown useful in the treatment of cell proliferative disorders including,for example, small cell lung, breast, cervical, head and neck, prostate,retinoblastoma, and soft tissue sarcoma.

The cell cycle inhibitor of the present invention may be a hydroxyurea.Hydroxyureas have the following general structure of formula (LXI):

Suitable hydroxyureas are disclosed in, for example, U.S. Pat. No.6,080,874, wherein R₁ is a group represented by the structure of formula(LXII):

wherein R₂ is an alkyl group having 1-4 carbons and R₃ is one of H,acyl, methyl, ethyl, and mixtures thereof, such as a methylether.

Other suitable hydroxyureas are disclosed in, e.g., U.S. Pat. No.5,665,768, wherein R₁ is a cycloalkenyl group, for exampleN-(3-(5-(4-fluorophenylthio)-furyl)-2-cyclopenten-1-yl)N-hydroxyurea; R₂is H or an alkyl group having 1 to 4 carbons and R₃ is H; X is H or acation.

Other suitable hydroxyureas are disclosed in, e.g., U.S. Pat. No.4,299,778, wherein R₁ is a phenyl group substituted with on or morefluorine atoms; R₂ is a cyclopropyl group; and R₃ and X is H.

Other suitable hydroxyureas are disclosed in, e.g., U.S. Pat. No.5,066,658, wherein R₂ and R₃ together with the adjacent nitrogen formwhich is represented by the structure of formula (XLIII):

wherein m is 1 or 2, n is 0-2 and Y is an alkyl group.

In one aspect, the hydroxy urea has the structure of formula (XLIV):

Hydroxyureas are thought to function as cell cycle inhibitors by servingto inhibit DNA synthesis.

In another aspect, the cell cycle inhibitor is a mytomicin, such asmitomycin C, or an analogue or derivative thereof, such asporphyromycin. Exemplary compounds have the structures of formula (XLV):

(XLV)

R Mitomycin C H Porphyromycin CH₃ (N-methyl Mitomycin C)

These compounds are thought to function as cell cycle inhibitors byserving as DNA alkylating agents. Mitomycins have been shown useful inthe treatment of cell proliferative disorders such as, for example,esophageal, liver, bladder, and breast cancers.

In another aspect, the cell cycle inhibitor is an alkyl sulfonate, suchas busulfan, or an analogue or derivative thereof, such as treosulfan,improsulfan, piposulfan, and pipobroman. Exemplary compounds have thefollowing structures:

R Busulfan single bond Improsulfan —CH₂—NH—CH₂— Piposulfan

These compounds are thought to function as cell cycle inhibitors byserving as DNA alkylating agents.

In another aspect, the cell cycle inhibitor is a benzamide. In yetanother aspect, the cell cycle inhibitor is a nicotinamide. Thesecompounds have the basic structure of formula (XLIII):

wherein X is either O or S; A is commonly NH₂ or it can be OH or analkoxy group; B is N or C—R₄, where R₄ is H or an ether-linkedhydroxylated alkane such as OCH₂CH₂OH, the alkane may be linear orbranched and may contain one or more hydroxyl groups. Alternately, B maybe N—R₅ in which case the double bond in the ring involving B is asingle bond. R₅ may be H, and alkyl or an aryl group (see, e.g., U.S.Pat. No. 4,258,052); R₂ is H, OR₆, SR₆, or NHR₆, where R₆ is an alkylgroup; and R₃ is H, a lower alkyl, an ether linked lower alkyl such as—O-Me or —O-ethyl (see, e.g., U.S. Pat. No. 5,215,738).

Suitable benzamide compounds have the structures of formula (XLIV):

wherein additional compounds are disclosed in U.S. Pat. No. 5,215,738,(listing some 32 compounds).

Suitable nicotinamide compounds have the structures of formula (XLV):

wherein additional compounds are disclosed in U.S. Pat. No. 5,215,738,such as:

In another aspect, the cell cycle inhibitor is a halogenated sugar, suchas mitolactol, or an analogue or derivative thereof, includingmitobronitol and mannomustine. Exemplary compounds have the structures:

In another aspect, the cell cycle inhibitor is a diazo compound, such asazaserine, or an analogue or derivative thereof, including6-diazo-5-oxo-L-norleucine and 5-diazouracil (also a pyrimidine analog).Exemplary compounds have the structures of formula (XLVI):

(XLVI)

R₁ R₂ Azaserine O single bond 6-diazo-5-oxo-L-norleucine single bond CH₂

Other compounds that may serve as cell cycle inhibitors according to thepresent invention are pazelliptine; wortmannin; metoclopramide; RSU;buthionine sulfoxime; tumeric; curcumin; AG337, a thymidylate synthaseinhibitor; levamisole; lentinan, a polysaccharide; razoxane, an EDTAanalogue; indomethacin; chlorpromazine; α and β interferon; MnBOPP;gadolinium texaphyrin; 4-amino-1,8-naphthalimide; staurosporinederivative of CGP; and SR-2508.

Thus, in one aspect, the cell cycle inhibitor is a DNA alylating agent.In another aspect, the cell cycle inhibitor is an anti-microtubuleagent. In another aspect, the cell cycle inhibitor is a topoisomeraseinhibitor. In another aspect, the cell cycle inhibitor is a DNA cleavingagent. In another aspect, the cell cycle inhibitor is an antimetabolite.In another aspect, the cell cycle inhibitor functions by inhibitingadenosine deaminase (e.g., as a purine analogue). In another aspect, thecell cycle inhibitor functions by inhibiting purine ring synthesisand/or as a nucleotide interconversion inhibitor (e.g., as a purineanalogue such as mercaptopurine). In another aspect, the cell cycleinhibitor functions by inhibiting dihydrofolate reduction and/or as athymidine monophosphate block (e.g., methotrexate). In another aspect,the cell cycle inhibitor functions by causing DNA damage (e.g.,bleomycin). In another aspect, the cell cycle inhibitor functions as aDNA intercalation agent and/or RNA synthesis inhibition (e.g.,doxorubicin, aclarubicin, or detorubicin (acetic acid, diethoxy-,2-[4-[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-2-naphthacenyl]-2-oxoethylester, (2S-cis)-)). In another aspect, the cell cycle inhibitorfunctions by inhibiting pyrimidine synthesis (e.g.,N-phosphonoacetyl-L-aspartate). In another aspect, the cell cycleinhibitor functions by inhibiting ribonucleotides (e.g., hydroxyurea).In another aspect, the cell cycle inhibitor functions by inhibitingthymidine monophosphate (e.g., 5-fluorouracil). In another aspect, thecell cycle inhibitor functions by inhibiting DNA synthesis (e.g.,cytarabine). In another aspect, the cell cycle inhibitor functions bycausing DNA adduct formation (e.g., platinum compounds). In anotheraspect, the cell cycle inhibitor functions by inhibiting proteinsynthesis (e.g., L-asparginase). In another aspect, the cell cycleinhibitor functions by inhibiting microtubule function (e.g., taxanes).In another aspect, the cell cycle inhibitor acts at one or more of thesteps in the biological pathway shown in FIG. 1.

Additional cell cycle inhibitor s useful in the present invention, aswell as a discussion of the mechanisms of action, may be found inHardman J. G., Limbird L. E. Molinoff R. B., Ruddon R W., Gilman A. G.editors, Chemotherapy of Neoplastic Diseases in Goodman and Gilman's ThePharmacological Basis of Therapeutics Ninth Edition, McGraw-Hill HealthProfessions Division, New York, 1996, pages 1225-1287. See also U.S.Pat. Nos. 3,387,001; 3,808,297; 3,894,000; 3,991,045; 4,012,390;4,057,548; 4,086,417; 4,144,237; 4,150,146; 4,210,584; 4,215,062;4,250,189; 4,258,052; 4,259,242; 4,296,105; 4,299,778; 4,367,239;4,374,414; 4,375,432; 4,472,379; 4,588,831; 4,639,456; 4,767,855;4,828,831; 4,841,045; 4,841,085; 4,908,356; 4,923,876; 5,030,620;5,034,320; 5,047,528; 5,066,658; 5,166,149; 5,190,929; 5,215,738;5,292,731; 5,380,897; 5,382,582; 5,409,915; 5,440,056; 5,446,139;5,472,956; 5,527,905; 5,552,156; 5,594,158; 5,602,140; 5,665,768;5,843,903; 6,080,874; 6,096,923; and RE030561.

In another embodiment, the cell-cycle inhibitor is camptothecin,mitoxantrone, etoposide, 5-fluorouracil, doxorubicin, methotrexate,peloruside A, mitomycin C, or a CDK-2 inhibitor or an analogue orderivative of any member of the class of listed compounds.

In another embodiment, the cell-cycle inhibitor is HTI-286, plicamycin;or mithramycin, or an analogue or derivative thereof.

Other examples of cell cycle inhibitors also include, e.g.,7-hexanoyltaxol (QP-2), cytochalasin A, lantrunculin D, actinomycin-D,Ro-31-7453(3-(6-nitro-1-methyl-3-indolyl)-4-(1-methyl-3-indolyl)pyrrole-2,5-dione),PNU-151807, brostallicin, C2-ceramide, cytarabine ocfosfate(2(1H)-pyrimidinone,4-amino-1-(5-O-(hydroxy(octadecyloxy)phosphinyl)-β-D-arabinofuranosyl)-,monosodium salt), paclitaxel (β,20-epoxy-1,2 alpha,4,7β,10β,13alpha-hexahydroxytax-11-en-9-one-4,10-diacetate-2-benzoate-13-(alpha-phenylhippurate)),doxorubicin (5,12-naphthacenedione,10-((3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-,(8S)-cis-), daunorubicin (5,12-naphthacenedione,8-acetyl-10-((3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-,(8S-cis)-), gemcitabine hydrochloride (cytidine,2′-deoxy-2′,2′-difluoro-,monohydrochloride), nitacrine(1,3-propanediamine, N,N-dimethyl-N′-(1-nitro-9-acridinyl)-),carboplatin (platinum, diammine(1,1-cyclobutanedicarboxylato(2-))-,(SP-4-2)-), altretamine (1,3,5-triazine-2,4,6-triamine,N,N,N′,N′,N″,N″-hexamethyl-), teniposide(furo(3′4,′:6,7)naphtho(2,3-d)-1,3-dioxol-6(5aH)-one,5,8,8a,9-tetrahydro-5-(4-hydroxy-3,5-dimethoxyphenyl)-9-(4,6-O-(2-thienylmethylene)-β-D-glucopyranosyl)oxy)-,(5R-(5alpha,5aβ,8aAlpha,9β(R*)))—), eptaplatin (platinum,((4R,5R)-2-(1-methylethyl)-1,3-dioxolane-4,5-dimethanamine-kappaN4,kappa N5)(propanedioato(2-)-kappa O1, kappa O3)-, (SP-4-2)-),amrubicin hydrochloride (5,12-naphthacenedione,9-acetyl-9-amino-7-((2-deoxy-β-D-erythro-pentopyranosyl)oxy)-7,8,9,10-tetrahydro-6,11-dihydroxy-,hydrochloride, (7S-cis)-), ifosfamide (2H-1,3,2-oxazaphosphorin-2-amine,N,3-bis(2-chloroethyl)tetrahydro-,2-oxide), cladribine (adenosine,2-chloro-2′-deoxy-), mitobronitol (D-mannitol,1,6-dibromo-1,6-dideoxy-), fludaribine phosphate (9H-purin-6-amine,2-fluoro-9-(5-O-phosphono-β-D-arabinofuranosyl)-), enocitabine(docosanamide,N-(1-β-D-arabinofuranosyl-1,2-dihydro-2-oxo-4-pyrimidinyl)-), vindesine(vincaleukoblastine,3-(aminocarbonyl)-O4-deacetyl-3-de(methoxycarbonyl)-), idarubicin(5,12-naphthacenedione,9-acetyl-7-((3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,9,11-trihydroxy-,(7S-cis)-), zinostatin (neocarzinostatin), vincristine(vincaleukoblastine, 22-oxo-), tegafur (2,4(1H,3H)-pyrimidinedione,5-fluoro-1-(tetrahydro-2-furanyl)-), razoxane (2,6-piperazinedione,4,4′-(1-methyl-1,2-ethanediyl)bis-), methotrexate (L-glutamic acid,N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-),raltitrexed (L-glutamic acid,N-((5-(((1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-),oxaliplatin (platinum,(1,2-cyclohexanediamine-N,N′)(ethanedioato(2-)—O,O′)-,(SP-4-2-(1R-trans))-), doxifluridine (uridine, 5′-deoxy-5-fluoro-),mitolactol (galactitol, 1,6-dibromo-1,6-dideoxy-), piraubicin(5,12-naphthacenedione,10-((3-amino-2,3,6-trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-,(8S-(8 alpha, 10 alpha(S*)))—), docetaxel((2R,3S)—N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with513,20-epoxy-1,2 alpha,4,7β,10β,13 alpha-hexahydroxytax-11-en-9-one4-acetate 2-benzoate-), capecitabine (cytidine,5-deoxy-5-fluoro-N-((pentyloxy)carbonyl)-), cytarabine(2(1H)-pyrimidone, 4-amino-1-β-D-arabino furanosyl-), valrubicin(pentanoic acid,2-(1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-4-((2,3,6-trideoxy-3-((trifluoroacetyl)amino)-alpha-L-lyxo-hexopyranosyl)oxy)-2-naphthacenyl)-2-oxoethylester (2S-cis)-), trofosfamide(3-2-(chloroethyl)-2-(bis(2-chloroethyl)amino)tetrahydro-2H-1,3,2-oxazaphosphorin2-oxide), prednimustine (pregna-1,4-diene-3,20-dione,21-(4-(4-(bis(2-chloroethyl)amino)phenyl)-1-oxobutoxy)-11,17-dihydroxy-,(11β)-), lomustine (Urea, N-(2-chloroethyl)-N′-cyclohexyl-N-nitroso-),epirubicin (5,12-naphthacenedione,10-((3-amino-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-,(8S-cis)-), or an analogue or derivative thereof).

Cyclin Dependent Protein Kinase Inhibitors

In another embodiment, the pharmacologically active compound is a cyclindependent protein kinase inhibitor (e.g., R-roscovitine, CYC-101,CYC-103, CYC-400, MX-7065, alvocidib (4H-1-Benzopyran-4-one,2-(2-chlorophenyl)-5,7-dihydroxy-8-(3-hydroxy-1-methyl-4-piperidinyl)-,cis-(−)-), SU-9516, AG-12275, PD-0166285, CGP-79807, fascaplysin,GW-8510 (benzenesulfonamide,4-(((Z)-(6,7-dihydro-7-oxo-8H-pyrrolo[2,3-g]benzothiazol-8-ylidene)methyl)amino)-N-(3-hydroxy-2,2-dimethylpropyl)-),GW-491619, Indirubin 3′ monoxime, GW8510, AZD-5438, ZK-CDK or ananalogue or derivative thereof).

EGF (Epidermal Growth Factor) Receptor Kinase Inhibitors

In another embodiment, the pharmacologically active compound is an EGF(epidermal growth factor) kinase inhibitor (e.g., erlotinib(4-quinazolinamine, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-,monohydrochloride), erbstatin, BIBX-1382, gefitinib (4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(4-morpholinyl)propoxy)), oran analogue or derivative thereof).

Elastase Inhibitors

In another embodiment, the pharmacologically active compound is anelastase inhibitor (e.g., ONO-6818, sivelestat sodium hydrate (glycine,N-(2-(((4-(2,2-dimethyl-1-oxopropoxy)phenyl)sulfonyl)amino)benzoyl)-),erdosteine (acetic acid,((2-oxo-2-((tetrahydro-2-oxo-3-thienyl)amino)ethyl)thio)-), MDL-100948A,MDL-104238(N-(4-(4-morpholinylcarbonyl)benzoyl)-L-valyl-N′-(3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl)-L-2-azetamide),MDL-27324 (L-prolinamide,N-((5-(dimethylamino)-1-naphthalenyl)sulfonyl)-L-alanyl-L-alanyl-N-(3,3,3-trifluoro-1-(1-methylethyl)-2-oxopropyl)-,(S)—), SR-26831 (thieno(3,2-c)pyridinium,542-chlorophenyl)methyl)-2-(2,2-dimethyl-1-oxopropoxy)-4,5,6,7-tetrahydro-5-hydroxy-),Win-68794, Win-63110, SSR-69071(2-(9(2-piperidinoethoxy)-4-oxo-4H-pyrido(1,2-a)pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methyoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide),(N(Alpha)-(1-adamantylsulfonyl)N(epsilon)-succinyl-L-lysyl-L-prolyl-L-valinal),Ro-31-3537 (Nalpha-(1-adamantanesulphonyl)-N-(4-carboxybenzoyl)-L-lysyl-alanyl-L-valinal),R-665, FCE-28204,((6R,7R)-2-(benzoyloxy)-7-methoxy-3-methyl-4-pivaloyl-3-cephem1,1-dioxide), 1,2-benzisothiazol-3(2H)-one, 2-(2,4-dinitrophenyl)-,1,1-dioxide, L-658758 (L-proline,143-((acetyloxy)methyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo(4.2.0)oct-2-en-2-yl)carbonyl)-,S,S-dioxide, (6R-cis)-), L-659286 (pyrrolidine,1-((7-methoxy-8-oxo-3-(((1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio)methyl)-5-thia-1-azabicyclo(4.2.0)oct-2-en-2-yl)carbonyl)-,S,S-dioxide, (6R-cis)-), L-680833 (benzeneacetic acid,4-((3,3-diethyl-1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-4-oxo-2-azetidinyl)oxy)-,(S—(R*,S*))-), FK-706 (L-prolinamide,N-[4-[[(carboxymethyl)amino]carbonyl]benzoyl]-L-valyl-N-[3,3,3-trifluoro-1-(1-methylethyl)-2-oxopropyl]-,monosodium salt), Roche R-665, or an analogue or derivative thereof).

Factor Xa Inhibitors

In another embodiment, the pharmacologically active compound is a factorXa inhibitor (e.g., CY-222, fondaparinux sodium(alpha-D-glucopyranoside, methylO-2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranosyl-(1-4)-O-β-D-glucopyranuronosyl-(1-4)-O-2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranosyl-(1-4)-O-2-O-sulfo-alpha-L-idopyranuronosyl-(1-4)-2-deoxy-2-(sulfoamino)-,6-(hydrogen sulfate)), danaparoid sodium, or an analogue or derivativethereof).

Farnesyltransferase Inhibitors

In another embodiment, the pharmacologically active compound is afarnesyltransferase inhibitor (e.g., dichlorobenzoprim(2,4-diamino-5-(4-(3,4-dichlorobenzylamino)-3-nitrophenyl)-6-ethylpyrimidine),B-581, B-956(N-(8(R)-amino-2(S)-benzyl-5(S)-isopropyl-9-sulfanyl-3(Z),6(E)-nonadienoyl)-L-methionine),OSI-754, perillyl alcohol (1-cyclohexene-1-methanol,4-(1-methylethenyl)-, RPR-114334, lonafarnib (1-piperidinecarboxamide,4-(2-(4-((11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo(5,6)cyclohepta(1,2-b)pyridin-11-yl)-1-piperidinyl)-2-oxoethyl)-),Sch-48755, Sch-226374,(7,8-dichloro-5H-dibenzo(b,e)(1,4)diazepin-11-yl)-pyridin-3-ylmethylamine,J-104126, L-639749, L-731734 (pentanamide,2-((2-(2-amino-3-mercaptopropyl)amino)-3-methylpentyl)amino)-3-methyl-N-(tetrahydro-2-oxo-3-furanyl)-,(3S-(3R*(2R*(2R*(S*),3S*),3R*)))-), L-744832 (butanoic acid,2-((2-((2-((2-amino-3-mercaptopropyl)amino)-3-methylpentyl)oxy)-1-oxo-3-phenylpropyl)amino)-4-(methylsulfonyl)-,1-methylethyl ester, (2S-(1(R*(R*)),2R*(S*),3R*))-), L-745631(1-piperazinepropanethiol,β-amino-2-(2-methoxyethyl)-4-(1-naphthalenylcarbonyl)-, (βR,2S)—),N-acetyl-N-naphthylmethyl-2(S)-((1-(4-cyanobenzyl)-1H-imidazol-5-yl)acetyl)amino-3(S)-methylpentamine,(2alpha)-2-hydroxy-24,25-dihydroxylanost-8-en-3-one, BMS-316810, UCF-1-C(2,4-decadienamide,N-(5-hydroxy-5-(7-((2-hydroxy-5-oxo-1-cyclopenten-1-yl)amino-oxo-1,3,5-heptatrienyl)-2-oxo-7-oxabicyclo(4.1.0)hept-3-en-3-yl)-2,4,6-trimethyl-,(1S-(1alpha,3(2E,4E,6S*),5 alpha, 5(1E,3E,5E), 6 alpha))-), UCF-116-B,ARGLABIN (3H-oxireno[8,8a]azuleno[4,5-b]furan-8(4aH)-one,5,6,6a,7,9a,9b-hexahydro-1,4a-dimethyl-7-methylene-, (3aR,4aS,6aS,9aS,9bR)—) from ARGLABIN-Paracure, Inc. (Virginia Beach,Va.), or an analogue or derivative thereof).

Fibrinogen Antagonists

In another embodiment, the pharmacologically active compound is afibrinogen antagonist (e.g.,2(S)-((p-toluenesulfonyl)amino)-3-(5,6,7,8,-tetrahydro-4-oxo-5-(2-(piperidin-4-yl)ethyl)-4H-pyrazolo-(1,5-a)(1,4)diazepin-2-yl)carbonyl)-amino)propionicacid, streptokinase (kinase (enzyme-activating), strepto-), urokinase(kinase (enzyme-activating), uro-), plasminogen activator, pamiteplase,monteplase, heberkinase, anistreplase, alteplase, pro-urokinase,picotamide (1,3-benzenedicarboxamide,4-methoxy-N,N′-bis(3-pyridinylmethyl)-), or an analogue or derivativethereof).

Guanylate Cyclase Stimulants

In another embodiment, the pharmacologically active compound is aguanylate cyclase stimulant (e.g., isosorbide-5-mononitrate (D-glucitol,1,4:3,6-dianhydro-, 5-nitrate), or an analogue or derivative thereof).

Heat Shock Protein 90 Antagonists

In another embodiment, the pharmacologically active compound is a heatshock protein 90 antagonist (e.g., geldanamycin; NSC-33050(17-allylaminogeldanamycin; 17-AAG), rifabutin (rifamycin XIV,1′,4-didehydro-1-deoxy-1,4-dihydro-5′-(2-methylpropyl)-1-oxo-), 17-DMAG,or an analogue or derivative thereof).

HMGCoA reductase inhibitors

In another embodiment, the pharmacologically active compound is anHMGCoA reductase inhibitor (e.g., BCP-671, BB-476, fluvastatin(6-heptenoic acid,7-(3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl)-3,5-dihydroxy-,monosodium salt, (R*,S*-(E))-(±)-), dalvastatin (2H-pyran-2-one,6-(2-(2-(2-(4-fluoro-3-methylphenyl)-4,4,6,6-tetramethyl-1-cyclohexen-1-yl)ethenyl)tetrahydro)-4-hydroxy-,(4alpha,613(E))-(+/−)-), glenvastatin (2H-pyran-2-one,6-(2-(4-(4-fluorophenyl)-2-(1-methylethyl)-6-phenyl-3-pyridinyl)ethenyl)tetrahydro-4-hydroxy-,(4R-(4alpha,6β(E)))-), S-2468,N-(1-oxododecyl)-4Alpha,10-dimethyl-8-aza-trans-decal-3β-ol,atorvastatin calcium (1H-Pyrrole-1-heptanoic acid,2-(4-fluorophenyl)-β,delta-dihydroxy-5-(1-methylethyl)-3-phenyl-4-((phenylamino)carbonyl)-,calcium salt (R—(R*,R*))-), CP-83101 (6,8-nonadienoic acid,3,5-dihydroxy-9,9-diphenyl-, methyl ester, (R*,S*-(E))-(+/−)-),pravastatin (1-naphthaleneheptanoic acid,1,2,6,7,8,8a-hexahydro-β,delta,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-,monosodium salt, (1S-(1 alpha(βS*,deltaS*),2 alpha,6 alpha,8β(R*),8aalpha))-), U-20685, pitavastatin (6-heptenoic acid,7-(2-cyclopropyl-4-(4-fluorophenyl)-3-quinolinyl)-3,5-dihydroxy-,calcium salt (2:1), (S—(R*,S*-(E)))-),N-((1-methylpropyl)carbonyl)-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-perhydro-isoquinoline,dihydromevinolin (butanoic acid, 2-methyl-,1,2,3,4,4a,7,8,8a-octahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-1-naphthalenylester(1 alpha(R*), 3 alpha, 4a alpha,7β,8β(2S*,4S*),8aβ))-), HBS-107,dihydromevinolin (butanoic acid, 2-methyl-,1,2,3,4,4a,7,8,8a-octahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-1-naphthalenylester(1 alpha(R*), 3 alpha,4a alpha,7β,8β(2S*,4S*),8aβ))-), L-669262(butanoic acid, 2,2-dimethyl-,1,2,6,7,8,8a-hexahydro-3,7-dimethyl-6-oxo-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-1-naphthalenyl(1S-(1Alpha,7β,8β(2S*,4S*),8aβ))-),simvastatin (butanoic acid, 2,2-dimethyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-1-naphthalenylester, (1S-(1alpha, 3alpha,7β,8β(2S*,4S*),8aβ))-), rosuvastatin calcium(6-heptenoic acid,7-(4-(4-fluorophenyl)-6-(1-methylethyl)-2-(methyl(methylsulfonyl)amino)-5-pyrimdinyl)-3,5-dihydroxy-calciumsalt (2:1) (5-(R*, S*-(E)))),meglutol(2-hydroxy-2-methyl-1,3-propandicarboxylic acid), lovastatin(butanoic acid, 2-methyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-(2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl)-1-naphthalenylester, (1S-(1 alpha.(R*),3 alpha,7β,8β(2S*,4S*),8aβ))-), or an analogueor derivative thereof).

Hydroorotate Dehydrogenase Inhibitors

In another embodiment, the pharmacologically active compound is ahydroorotate dehydrogenase inhibitor (e.g., leflunomide(4-isoxazolecarboxamide, 5-methyl-N-(4-(trifluoromethyl)phenyl)-),laflunimus (2-propenamide,2-cyano-3-cyclopropyl-3-hydroxy-N-(3-methyl-4(trifluoromethyl)phenyl)-,(Z)—), or atovaquone (1,4-naphthalenedione,2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-, trans-, or an analogue orderivative thereof).

IKK2 Inhibitors

In another embodiment, the pharmacologically active compound is an IKK2inhibitor (e.g., MLN-120B, SPC-839, or an analogue or derivativethereof).

IL-1, ICE and IRAK Antagonists

In another embodiment, the pharmacologically active compound is an IL-1,ICE or an IRAK antagonist (e.g., E-5090 (2-propenoic acid,3-(5-ethyl-4-hydroxy-3-methoxy-1-naphthalenyl)-2-methyl-, (Z)—), CH-164,CH-172, CH-490, AMG-719, iguratimod(N-(3-(formylamino)-4-oxo-6-phenoxy-4H-chromen-7-yl)methanesulfonamide), AV94-88, pralnacasan(6H-pyridazino(1,2-a)(1,2)diazepine-1-carboxamide,N-((2R,3S)-2-ethoxytetrahydro-5-oxo-3-furanyl)octahydro-9-((1-isoquinolinylcarbonyl)amino)-6,10-dioxo-,(1S,9S)—),(25-cis)-5-(benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-(oxoazepino(3,2,1-hi)indole-2-carbonyl)-amino)-4-oxobutanoicacid, AVE-9488, esonarimod (benzenebutanoic acid,alpha-((acetylthio)methyl)-4-methyl-γ-oxo-), pralnacasan(6H-pyridazino(1,2-a)(1,2)diazepine-1-carboxamide,N-((2R,3S)-2-ethoxytetrahydro-5-oxo-3-furanyl)octahydro-9-((1-isoquinolinylcarbonyl)amino)-6,10-dioxo-,(1S,9S)—), tranexamic acid (cyclohexanecarboxylic acid,4-(aminomethyl)-, trans-), Win-72052, romazarit (Ro-31-3948) (propanoicacid, 2-((2-(4-chlorophenyl)-4-methyl-5-oxazolyl)methoxy)-2-methyl-),PD-163594, SDZ-224-015 (L-alaninamideN-((phenylmethoxy)carbonyl)-L-valyl-N-((1S)-3-((2,6-dichlorobenzoyl)oxy)-1-(2-ethoxy-2-oxoethyl)-2-oxopropyl)-),L-709049 (L-alaninamide,N-acetyl-L-tyrosyl-L-valyl-N-(2-carboxy-1-formylethyl)-, (S)—), TA-383(1H-imidazole, 2-(4-chlorophenyl)-4,5-dihydro-4,5-diphenyl-,monohydrochloride, cis-), EI-1507-1(6a,12a-epoxybenz(a)anthracen-1,12(2H,7H)-dione,3,4-dihydro-3,7-dihydroxy-8-methoxy-3-methyl-), ethyl4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmethyl)quinoline-3-carboxylate, EI-1941-1, TJ-114, anakinra (interleukin1 receptor antagonist (human isoform x reduced), N2-L-methionyl-),IX-207-887 (acetic acid,(10-methoxy-4H-benzo[4,5]cyclohepta[1,2-b]thien-4-ylidene)-), K-832, oran analogue or derivative thereof).

IL-4 Agonists

In another embodiment, the pharmacologically active compound is an IL-4agonist (e.g., glatiramir acetate (L-glutamic acid, polymer withL-alanine, L-lysine and L-tyrosine, acetate (salt)), or an analogue orderivative thereof).

Immunomodulatory Agents

In another embodiment, the pharmacologically active compound is animmunomodulatory agent (e.g., biolimus, ABT-578, methylsulfamic acid3-(2-methoxyphenoxy)-2-(((methylamino)sulfonyl)oxy)propyl ester,sirolimus (also referred to as rapamycin or RAPAMUNE (American HomeProducts, Inc., Madison, N.J.)), CCI-779 (rapamycin42-(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate)), LF-15-0195,NPC15669 (L-leucine,N-(((2,7-dimethyl-9H-fluoren-9-yl)methoxy)carbonyl)-), NPC-15670(L-leucine, N-(((4,5-dimethyl-9H-fluoren-9-yl)methoxy)carbonyl)-),NPC-16570 (4-(2-(fluoren-9-yl)ethyloxy-carbonyl)aminobenzoic acid),sufosfamide (ethanol,2-((3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-yl)amino)-,methanesulfonate (ester), P-oxide), tresperimus(2-(N-(4-(3-aminopropylamino)butyl)carbamoyloxy)-N-(6-guanidinohexyl)acetamide),4-(2-(fluoren-9-yl)ethoxycarbonylamino)-benzo-hydroxamic acid,iaquinimod, PBI-1411, azathioprine(6-((1-Methyl-4-nitro-1H-imidazol-5-yl)thio)-1H-purine), PBI0032,beclometasone, MDL-28842 (9H-purin-6-amine,9-(5-deoxy-5-fluoro-β-D-threo-pent-4-enofuranosyl)-, (Z)—), FK-788,AVE-1726, ZK-90695, ZK-90695, Ro-54864, didemnin-B, Illinois (didemninA, N-(1-(2-hydroxy-1-oxopropyl)-L-prolyl)-, (S)—), SDZ-62-826(ethanaminium,2-((hydroxy((1-((octadecyloxy)carbonyl)-3-piperidinyl)methoxy)phosphinyl)oxy)-N,N,N-trimethyl-,inner salt), argyrin B((4S,7S,13R,22R)-13-Ethyl-4-(1H-indol-3-ylmethyl)-7-(4-methoxy-1H-indol-3-ylmethyl)18,22-dimethyl-16-methyl-ene-24-thia-3,6,9,12,15,18,21,26-octaazabicyclo(21.2.1)-hexacosa-1(25),23(26)-diene-2,5,8,11,14,17,20-heptaone), everolimus (rapamycin,42-O-(2-hydroxyethyl)-), SAR-943, L-687795,644-chlorophenyl)sulfinyl)-2,3-dihydro-2-(4-methoxy-phenyl)-5-methyl-3-oxo-4-pyridazinecarbonitrile,91Y78 (1H-imidazo[4,5-c]pyridin-4-amine, 1-β-D-ribofuranosyl-),auranofin (gold, (1-thio-β-D-glucopyranose2,3,4,6-tetraacetato-S)(triethylphosphine)-), 27-0-demethylrapamycin,tipredane (androsta-1,4-dien-3-one,17-(ethylthio)-9-fluoro-11-hydroxy-17-(methylthio)-, (11β,17 alpha)-),AI-402, LY-178002 (4-thiazolidinone,5-((3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl)methylene)-), SM-8849(2-thiazolamine, 4-(1-(2-fluoro(1,1′-biphenyl)-4-yl)ethyl)-N-methyl-),piceatannol, resveratrol, triamcinolone acetonide(pregna-1,4-diene-3,20-dione,9-fluoro-11,21-dihydroxy-16,17-((1-methylethylidene)bis(oxy))-, (11B,16alpha)-), ciclosporin (cyclosporin A), tacrolimus(15,19-epoxy-3H-pyrido(2,1-c)(1,4)oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone,5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl)-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-,(3S-(3R*(E(1S*,3S*,4S*)),4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*))-),gusperimus (heptanamide,7-((aminoiminomethyl)amino)-N-(2-((4-((3-aminopropyl)amino)butyl)amino)-1-hydroxy-2-oxoethyl)-,(+/−)-), tixocortol pivalate (pregn-4-ene-3,20-dione,21-((2,2-dimethyl-1-oxopropyl)thio)-11,17-dihydroxy-, (11β)-), alefacept(1-92 LFA-3 (antigen) (human) fusion protein with immunoglobulin G1(human hinge-CH2-CH3 gamma-1-chain), dimer), halobetasol propionate(pregna-1,4-diene-3,20-dione,21-chloro-6,9-difluoro-11-hydroxy-16-methyl-17-(1-oxopropoxy)-,(6Alpha,11β,16β)-), iloprost trometamol (pentanoic acid,5-(hexahydro-5-hydroxy-4-(3-hydroxy-4-methyl-1-octen-6-ynyl)-2(1H)-pentalenylidene)-),beraprost (1H-cyclopenta(b)benzofuran-5-butanoic acid,2,3,3a,8b-tetrahydro-2-hydroxy-1-(3-hydroxy-4-methyl-1-octen-6-ynyl)-),rimexolone(androsta-1,4-dien-3-one,11-hydroxy-16,17-dimethyl-17-(1-oxopropyl)-,(11β,16Alpha,17β)-), dexamethasone(pregna-1,4-diene-3,20-dione,9-fluoro-11,17,21-trihydroxy-16-methyl-,(11β,16alpha)-), sulindac(cis-5-fluoro-2-methyl-1-((p-methylsulfinyl)benzylidene)indene-3-aceticacid), proglumetacin (1H-Indole-3-acetic acid,1-(4-chlorobenzoyl)-5-methoxy-2-methyl-,2-(4-(3-((4-(benzoylamino)-5-(dipropylamino)-1,5-dioxopentyl)oxy)propyl)-1-piperazinyl)ethylester,(+/−)-), alclometasone dipropionate (pregna-1,4-diene-3,20-dione,7-chloro-1′-hydroxy-16-methyl-17,21-bis(1-oxopropoxy)-,(7alpha,11β,16alpha)-), pimecrolimus(15,19-epoxy-3H-pyrido(2,1-c)(1,4)oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone,3-(2-(4-chloro-3-methoxycyclohexyl)-1-methyletheny)-8-ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-14,16-dimethoxy-4,10,12,18-tetramethyl-,(3S-(3R*(E(1S*,3S*,4R*)),4S*,5R*,8S*,9E,12R*,14R*,155*,16R*,18S*,19S*,26aR*))-),hydrocortisone-17-butyrate (pregn-4-ene-3,20-dione,11,21-dihydroxy-17-(1-oxobutoxy)-, (11β)-), mitoxantrone(9,10-anthracenedione,1,4-dihydroxy-5,8-bis((2-((2-hydroxyethyl)amino)ethyl)amino)-),mizoribine (1H-imidazole-4-carboxamide, 5-hydroxy-1-β-D-ribofuranosyl-),prednicarbate (pregna-1,4-diene-3,20-dione,17-((ethoxycarbonyl)oxy)-11-hydroxy-21-(1-oxopropoxy)-, (11β)-),iobenzarit (benzoic acid, 2-((2-carboxyphenyl)amino)-4-chloro-),glucametacin (D-glucose,2-(((1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetyl)amino)-2-deoxy-),fluocortolone monohydrate ((6alpha)-fluoro-16alpha-methylpregna-1,4-dien-11β,21-diol-3,20-dione),fluocortin butyl (pregna-1,4-dien-21-oic acid,6-fluoro-11-hydroxy-16-methyl-3,20-dioxo-, butyl ester,(6alpha,11β,16alpha)-), difluprednate (pregna-1,4-diene-3,20-dione,21-(acetyloxy)-6,9-difluoro-11-hydroxy-17-(1-oxobutoxy)-, (6alpha,11β)-), diflorasone diacetate (pregna-1,4-diene-3,20-dione,17,21-bis(acetyloxy)-6,9-difluoro-11-hydroxy-16-methyl-,(6Alpha,11β,16β)-), dexamethasone valerate (pregna-1,4-diene-3,20-dione,9-fluoro-11,21-dihydroxy-16-methyl-17-((1-oxopentyl)oxy)-,(11β,16Alpha)-), methylprednisolone, deprodone propionate(pregna-1,4-diene-3,20-dione, 11-hydroxy-17-(1-oxopropoxy)-,(11.beta.)-), bucillamine (L-cysteine,N-(2-mercapto-2-methyl-1-oxopropyl)-), amcinonide (benzeneacetic acid,2-amino-3-benzoyl-, monosodium salt, monohydrate), acemetacin(1H-indole-3-acetic acid, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-,carboxymethyl ester), or an analogue or derivative thereof).

Further, analogues of rapamycin include tacrolimus and derivativesthereof (e.g., EP0184162B1 and U.S. Pat. No. 6,258,823) everolimus andderivatives thereof (e.g., U.S. Pat. No. 5,665,772). Furtherrepresentative examples of sirolimus analogues and derivatives can befound in PCT Publication Nos. WO 97/10502, WO 96/41807, WO 96/35423, WO96/03430, WO 96/00282, WO 95/16691, WO 95/15328, WO 95/07468, WO95/04738, WO 95/04060, WO 94/25022, WO 94/21644, WO 94/18207, WO94/10843, WO 94/09010, WO 94/04540, WO 94/02485, WO 94/02137, WO94/02136, WO 93/25533, WO 93/18043, WO 93/13663, WO 93/11130, WO93/10122, WO 93/04680, WO 92/14737, and WO 92/05179. Representative U.S.patents include U.S. Pat. Nos. 6,342,507; 5,985,890; 5,604,234;5,597,715; 5,583,139; 5,563,172; 5,561,228; 5,561,137; 5,541,193;5,541,189; 5,534,632; 5,527,907; 5,484,799; 5,457,194; 5,457,182;5,362,735; 5,324,644; 5,318,895; 5,310,903; 5,310,901; 5,258,389;5,252,732; 5,247,076; 5,225,403; 5,221,625; 5,210,030; 5,208,241;5,200,411; 5,198,421; 5,147,877; 5,140,018; 5,116,756; 5,109,112;5,093,338; and 5,091,389.

The structures of sirolimus, everolimus, and tacrolimus are providedbelow in Table 3:

TABLE 3 Name Code Name Company Structure Everolimus SAR-943 Novartis Seebelow Sirolimus AY-22989 Wyeth See below RAPAMUNE ® NSC-226080 RapamycinTacrolimus FK506 Fujusawa See below

Further sirolimus analogues and derivatives include tacrolimus andderivatives thereof (e.g., EP0184162B1 and U.S. Pat. No. 6,258,823)everolimus and derivatives thereof (e.g., U.S. Pat. No. 5,665,772).Further representative examples of sirolimus analogues and derivativesinclude ABT-578 and others may be found in PCT Publication Nos. WO97/10502, WO 96/41807, WO 96/35423, WO 96/03430, WO 9600282, WO95/16691, WO 95/5328, WO 95/07468, WO 95/04738, WO 95/04060, WO94/25022, WO 94/21644, WO 94/18207, WO 94/10843, WO 94/09010, WO94/04540, WO 94/02485, WO 94/02137, WO 94/02136, WO 93/25533, WO93/18043, WO 93/13663, WO 93/11130, WO 93/10122, WO 93/04680, WO92/14737, and WO 92/05179. Representative U.S. patents include U.S. Pat.Nos. 6,342,507; 5,985,890; 5,604,234; 5,597,715; 5,583,139; 5,563,172;5,561,228; 5,561,137; 5,541,193; 5,541,189; 5,534,632; 5,527,907;5,484,799; 5,457,194; 5,457,182; 5,362,735; 5,324,644; 5,318,895;5,310,903; 5,310,901; 5,258,389; 5,252,732; 5,247,076; 5,225,403;5,221,625; 5,210,030; 5,208,241, 5,200,411; 5,198,421; 5,147,877;5,140,018; 5,116,756; 5,109,112; 5,093,338; and 5,091,389.

In one aspect, the fibrosis-inhibiting agent may be, e.g., rapamycin(sirolimus), everolimus, biolimus, tresperimus, auranofin,27-0-demethylrapamycin, tacrolimus, gusperimus, pimecrolimus, orABT-578.

Inosine Monophosphate Dehydrogenase Inhibitors

In another embodiment, the pharmacologically active compound is aninosine monophosphate dehydrogenase (IMPDH) inhibitor (e.g.,mycophenolic acid, mycophenolate mofetil (4-hexenoic acid,6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-,2-(4-morpholinyl)ethyl ester, (E)-), ribavirin(1H-1,2,4-triazole-3-carboxamide, 1-β-D-ribofuranosyl-), tiazofurin(4-thiazolecarboxamide, 2-β-D-ribofuranosyl-), viramidine,aminothiadiazole, thiophenfurin, tiazofurin) or an analogue orderivative thereof. Additional representative examples are included inU.S. Pat. Nos. 5,536,747, 5,807,876, 5,932,600, 6,054,472, 6,128,582,6,344,465, 6,395,763, 6,399,773, 6,420,403, 6,479,628, 6,498,178,6,514,979, 6,518,291, 6,541,496, 6,596,747, 6,617,323, 6,624,184, PatentApplication Publication Nos. 2002/0040022A1, 2002/0052513A1,2002/0055483A1, 2002/0068346A1, 2002/0111378A1, 2002/0111495A1,2002/0123520A1, 2002/0143176A1, 2002/0147160A1, 2002/0161038A1,2002/0173491A1, 2002/0183315A1, 2002/0193612A1, 2003/0027845A1,2003/0068302A1, 2003/0105073A1, 2003/0130254A1, 2003/0143197A1,2003/0144300A1, 2003/0166201A1, 2003/0181497A1, 2003/0186974A1,2003/0186989A1, 2003/0195202A1, and PCT Publication Nos. WO 0024725A1,WO 00/25780A1, WO 00/26197A1, WO 00/51615A1, WO 00/56331A1, WO00/73288A1, WO 01/00622A1, WO 01/66706A1, WO 01/79246A2, WO 01/81340A2,WO 01/85952A2, WO 02/16382A1, WO 02/18369A2, WO 2051814A1, WO 2057287A2,WO2057425A2, WO 2060875A1, WO 2060896A1, WO 2060898A1, WO 2068058A2, WO3020298A1, WO 3037349A1, WO 3039548A1, WO 3045901A2, WO 3047512A2, WO3053958A1, WO 3055447A2, WO 3059269A2, WO 3063573A2, WO 3087071A1, WO90/01545A1, WO 97/40028A1, WO 97/41211A1, WO 98/40381A1, and WO99/55663A1).

Leukotriene Inhibitors

In another embodiment, the pharmacologically active compound is aleukotriene inhibitor (e.g., ONO-4057(benzenepropanoic acid,2-(4-carboxybutoxy)-6-((6-(4-methoxyphenyl)-5-hexenyl)oxy)-, (E)-),ONO-LB-448, pirodomast 1,8-naphthyridin-2(1H)-one,4-hydroxy-1-phenyl-3-(1-pyrrolidinyl)-, Sch-40120(benzo(b)(1,8)naphthyridin-5(7H)-one,10-(3-chlorophenyl)-6,8,9,10-tetrahydro-), L-656224 (4-benzofuranol,7-chloro-2((4-methoxyphenyl)methyl)-3-methyl-5-propyl-), MAFP (methylarachidonyl fluorophosphonate), ontazolast (2-benzoxazolamine,N-(2-cyclohexyl-1-(2-pyridinyl)ethyl)-5-methyl-, (S)—), amelubant(carbamic acid,((4-((3-((4-(1-(4-hydroxyphenyl)-1-methylethyl)phenoxy)methyl)phenyl)methoxy)phenyl)iminomethyl)-ethylester), SB-201993 (benzoic acid,3-((((6-((1E)-2-carboxyethenyl)-548-(4-methoxyphenyl)octyl)oxy)-2-pyridinyl)methyl)thio)methyl)-),LY-203647 (ethanone,1-(2-hydroxy-3-propyl-4-(4-(2-(4-(1H-tetrazol-5-yl)butyl)-2H-tetrazol-5-yl)butoxy)phenyl)-),LY-210073, LY-223982 (benzenepropanoic acid,5-(3-carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5-hexenyl)oxy)-, (E)-),LY-293111 (benzoic acid,2-(3-(3-((5-ethyl-4′-fluoro-2-hydroxy(1,1′-biphenyl)-4-yl)oxy)propoxy)-2-propylphenoxy)-),SM-9064 (pyrrolidine,1-(4,11-dihydroxy-13-(4-methoxyphenyl)-1-oxo-5,7,9-tridecatrienyl)-,(E,E,E)-), T-0757 (2,6-octadienamide,N-(4-hydroxy-3,5-dimethylphenyl)-3,7-dimethyl-, (2E)-), or an analogueor derivative thereof).

MCP-1 Antagonists

In another embodiment, the pharmacologically active compound is a MCP-1antagonist (e.g., nitronaproxen (2-napthaleneacetic acid,6-methoxy-alpha-methyl 4-(nitrooxy)butyl ester (alpha S)—), bindarit(2-(1-benzylindazol-3-ylmethoxy)-2-methylpropanoic acid), 1-alpha-25dihydroxy vitamin D₃, or an analogue or derivative thereof).

MMP Inhibitors

In another embodiment, the pharmacologically active compound is a matrixmetalloproteinase (MMP) inhibitor (e.g., D-9120, doxycycline(2-naphthacenecarboxamide,4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,1′-dioxo-(4S-(4alpha, 4a alpha, 5 lpha, 5a alpha, 6 alpha, 12a alpha))-), BB-2827,BB-1101(2S-allyl-N1-hydroxy-3R-isobutyl-N4-(1S-methylcarbamoyl-2-phenylethyl)-succinamide),BB-2983, solimastat(N′-(2,2-dimethyl-1(S)—(N-(2-pyridyl)carbamoyl)propyl)-N4-hydroxy-2(R)-isobutyl-3(S)-methoxysuccinamide),batimastat (butanediamide,N4-hydroxy-N1-(2-(methylamino)-2-oxo-1-(phenylmethyl)ethyl)-2-(2-methylpropyl)-3-((2-thienylthio)methyl)-,(2R-(1(S*),2R*,3S*))-), CH-138, CH-5902, D-1927, D-5410, EF-13(γ-linolenic acid lithium salt), CMT-3 (2-naphthacenecarboxamide,1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxo-,(4aS,5aR,12aS)—), marimastat(N-(2,2-dimethyl-1(S)—(N-methylcarbamoyl)propyl)-N,3(S)-dihydroxy-2(R)-isobutylsuccinamide),TIMP′S,ONO-4817, rebimastat (L-Valinamide,N-((2S)-2-mercapto-1-oxo-4-(3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl)butyl)-L-leucyl-N,3-dimethyl-),PS-508, CH-715, nimesulide (methanesulfonamide,N-(4-nitro-2-phenoxyphenyl)-),hexahydro-2-(2(R)-(1(RS)-(hydroxycarbamoyl)-4-phenylbutyl)nonanoyl)-N-(2,2,6,6-tetramethyl-4-piperidinyl)-3(S)-pyridazinecarboxamide, Rs-113-080, Ro-1130830, cipemastat (1-piperidinebutanamide,β-(cyclopentylmethyl)-N-hydroxy-γ-oxo-α-((3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl)methyl)-,(alphaR,βR)—), 5-(4′-biphenyl)-5-(N-(4-nitrophenyl)piperazinyl)barbituricacid, 6-methoxy-1,2,3,4-tetrahydro-norharman-1-carboxylic acid,Ro-31-4724 (L-alanine,N-(2-(2-(hydroxyamino)-2-oxoethyl)-4-methyl-1-oxopentyl)-L-leucyl-,ethyl ester), prinomastat (3-thiomorpholinecarboxamide,N-hydroxy-2,2-dimethyl-4-((4-(4-pyridinyloxy) phenyl)sulfonyl)-, (3R)—),AG-3433 (1H-pyrrole-3-propanic acid,1-(4′-cyano(1,1′-biphenyl)-4-yl)-b-((((3S)-tetrahydro-4,4-dimethyl-2-oxo-3-furanyl)amino)carbonyl)-,phenylmethyl ester, (bS)—), PNU-142769 (2H-Isoindole-2-butanamide,1,3-dihydro-N-hydroxy-alpha-((3S)-3-(2-methylpropyl)-2-oxo-1-(2-phenylethyl)-3-pyrrolidinyl)-1,3-dioxo-,(alpha R)—),(S)-1-(2-((((4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)amino)-carbonyl)amino)-1-oxo-3-(pentafluorophenyl)propyl)-4-(2-pyridinyl)piperazine,SU-5402 (1H-pyrrole-3-propanoic acid,2-((1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl)-4-methyl-), SC-77964,PNU-171829, CGS-27023A,N-hydroxy-2(R)-((4-methoxybenzene-sulfonyl)(4-picolyl)amino)-2-(2-tetrahydrofuranyl)-acetamide,L-758354 ((1,1′-biphenyl)-4-hexanoic acid,alpha-butyl-gamma-(((2,2-dimethyl-1-((methylamino)carbonyl)propyl)amino)carbonyl)-4′-fluoro-,(alpha S-(alpha R*, gammaS*(R*)))-, GI-155704A, CPA-926, TMI-005,XL-784, or an analogue or derivative thereof). Additional representativeexamples are included in U.S. Pat. Nos. 5,665,777; 5,985,911; 6,288,261;5,952,320; 6,441,189; 6,235,786; 6,294,573; 6,294,539; 6,563,002;6,071,903; 6,358,980; 5,852,213; 6,124,502; 6,160,132; 6,197,791;6,172,057; 6,288,086; 6,342,508; 6,228,869; 5,977,408; 5,929,097;6,498,167; 6,534,491; 6,548,524; 5,962,481; 6,197,795; 6,162,814;6,441,023; 6,444,704; 6,462,073; 6,162,821; 6,444,639; 6,262,080;6,486,193; 6,329,550; 6,544,980; 6,352,976; 5,968,795; 5,789,434;5,932,763; 6,500,847; 5,925,637; 6,225,314; 5,804,581; 5,863,915;5,859,047; 5,861,428; 5,886,043; 6,288,063; 5,939,583; 6,166,082;5,874,473; 5,886,022; 5,932,577; 5,854,277; 5,886,024; 6,495,565;6,642,255; 6,495,548; 6,479,502; 5,696,082; 5,700,838; 6,444,639;6,262,080; 6,486,193; 6,329,550; 6,544,980; 6,352,976; 5,968,795;5,789,434; 5,932,763; 6,500,847; 5,925,637; 6,225,314; 5,804,581;5,863,915; 5,859,047; 5,861,428; 5,886,043; 6,288,063; 5,939,583;6,166,082; 5,874,473; 5,886,022; 5,932,577; 5,854,277; 5,886,024;6,495,565; 6,642,255; 6,495,548; 6,479,502; 5,696,082; 5,700,838;5,861,436; 5,691,382; 5,763,621; 5,866,717; 5,902,791; 5,962,529;6,017,889; 6,022,873; 6,022,898; 6,103,739; 6,127,427; 6,258,851;6,310,084; 6,358,987; 5,872,152; 5,917,090; 6,124,329; 6,329,373;6,344,457; 5,698,706; 5,872,146; 5,853,623; 6,624,144; 6,462,042;5,981,491; 5,955,435; 6,090,840; 6,114,372; 6,566,384; 5,994,293;6,063,786; 6,469,020; 6,118,001; 6,187,924; 6,310,088; 5,994,312;6,180,611; 6,110,896; 6,380,253; 5,455,262; 5,470,834; 6,147,114;6,333,324; 6,489,324; 6,362,183; 6,372,758; 6,448,250; 6,492,367;6,380,258; 6,583,299; 5,239,078; 5,892,112; 5,773,438; 5,696,147;6,066,662; 6,600,057; 5,990,158; 5,731,293; 6,277,876; 6,521,606;6,168,807; 6,506,414; 6,620,813; 5,684,152; 6,451,791; 6,476,027;6,013,649; 6,503,892; 6,420,427; 6,300,514; 6,403,644; 6,177,466;6,569,899; 5,594,006; 6,417,229; 5,861,510; 6,156,798; 6,387,931;6,350,907; 6,090,852; 6,458,822; 6,509,337; 6,147,061; 6,114,568;6,118,016; 5,804,593; 5,847,153; 5,859,061; 6,194,451; 6,482,827;6,638,952; 5,677,282; 6,365,630; 6,130,254; 6,455,569; 6,057,369;6,576,628; 6,110,924; 6,472,396; 6,548,667; 5,618,844; 6,495,578;6,627,411; 5,514,716; 5,256,657; 5,773,428; 6,037,472; 6,579,890;5,932,595; 6,013,792; 6,420,415; 5,532,265; 5,691,381; 5,639,746;5,672,598; 5,830,915; 6,630,516; 5,324,634; 6,277,061; 6,140,099;6,455,570; 5,595,885; 6,093,398; 6,379,667; 5,641,636; 5,698,404;6,448,058; 6,008,220; 6,265,432; 6,169,103; 6,133,304; 6,541,521;6,624,196; 6,307,089; 6,239,288; 5,756,545; 6,020,366; 6,117,869;6,294,674; 6,037,361; 6,399,612; 6,495,568; 6,624,177; 5,948,780;6,620,835; 6,284,513; 5,977,141; 6,153,612; 6,297,247; 6,559,142;6,555,535; 6,350,885; 5,627,206; 5,665,764; 5,958,972; 6,420,408;6,492,422; 6,340,709; 6,022,948; 6,274,703; 6,294,694; 6,531,499;6,465,508; 6,437,177; 6,376,665; 5,268,384; 5,183,900; 5,189,178;6,511,993; 6,617,354; 6,331,563; 5,962,466; 5,861,427; 5,830,869; and6,087,359.

Nf Kappa B Inhibitors

In another embodiment, the pharmacologically active compound is a NFkappa B (NFKB) inhibitor (e.g., AVE-0545, Oxi-104 (benzamide,4-amino-3-chloro-N-(2-(diethylamino)ethyl)-), dexlipotam, R-flurbiprofen((1,1′-biphenyl)-4-acetic acid, 2-fluoro-alpha-methyl), SP100030(2-chloro-N-(3,5-di(trifluoromethyl)phenyl)-4-(trifluoromethyl)pyrimidine-5-carboxamide),AVE-0545, Viatris, AVE-0547, Bay 11-7082, Bay 11-7085, 15deoxy-prostaglandin J2, bortezomib (boronic acid,((1R)-3-methyl-1-(((2S)-1-oxo-3-phenyl-2-((pyrazinylcarbonyl)amino)propyl)amino)butyl)-,benzamide and nicotinamide derivatives that inhibit NF-kappaB, such asthose described in U.S. Pat. Nos. 5,561,161 and 5,340,565 (OxiGene),PG490-88Na, or an analogue or derivative thereof).

No Agonists

In another embodiment, the pharmacologically active compound is a NOantagonist (e.g., NCX-4016 (benzoic acid, 2-(acetyloxy)-,3-((nitrooxy)methyl)phenyl ester, NCX-2216, L-arginine or an analogue orderivative thereof).

P38 Map Kinase Inhibitors

In another embodiment, the pharmacologically active compound is a p38MAP kinase inhibitor (e.g., GW-2286, CGP-52411, BIRB-798, SB220025,RO-320-1195, RWJ-67657, RWJ-68354, SCIO-469, SCIO-323, AMG-548, CMC-146,SD-31145, CC-8866, Ro-320-1195, PD-98059 (4H-1-benzopyran-4-one,2-(2-amino-3-methoxyphenyl)-), CGH-2466, doramapimod, SB-203580(pyridine,44544-fluorophenyl)-2-(4-(methylsulfinyl)phenyl)-1H-imidazol-4-yl)-),SB-220025((5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole),SB-281832, PD169316, SB202190, GSK-681323, EO-1606, GSK-681323, or ananalogue or derivative thereof). Additional representative examples areincluded in U.S. Pat. Nos. 6,300,347; 6,316,464; 6,316,466; 6,376,527;6,444,696; 6,479,507; 6,509,361; 6,579,874; 6,630,485, U.S. PatentApplication Publication Nos. 2001/0044538A1; 2002/0013354A1;2002/0049220A1; 2002/0103245A1; 2002/0151491A1; 2002/0156114A1;2003/0018051A1; 2003/0073832A1; 2003/0130257A1; 2003/0130273A1;2003/0130319A1; 2003/0139388A1; 20030139462A1; 2003/0149031A1;2003/0166647A1; 2003/0181411A1; and PCT Publication Nos. WO 00/63204A2;WO 01/21591A1; WO 01/35959A1; WO 01/74811A2; WO 02/18379A2; WO2064594A2; WO 2083622A2; WO 2094842A2; WO 2096426A1; WO 2101015A2; WO2103000A2; WO 3008413A1; WO 3016248A2; WO 3020715A1; WO 3024899A2; WO3031431A1; WO3040103A1; WO 3053940A1; WO 3053941A2; WO 3063799A2; WO3079986A2; WO 3080024A2; WO 3082287A1; WO 97/44467A1; WO 99/01449A1; andWO 99/58523A1.

Phosphodiesterase Inhibitors

In another embodiment, the pharmacologically active compound is aphosphodiesterase inhibitor (e.g., CDP-840 (pyridine,4-((2R)-2-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-phenylethyl)-),CH-3697, CT-2820, D-22888 (imidazo[1,5-a]pyrido(3,2-e)pyrazin-6(5H)-one,9-ethyl-2-methoxy-7-methyl-5-propyl-), D-4418(8-methoxyquinoline-5-(N-(2,5-dichloropyridin-3-yl))carboxamide),1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(2,6-dichloro-4-pyridyl)ethanoneoxime, D-4396, ONO-6126, CDC-998, CDC-801, V-11294A(3-(3-(cyclopentyloxy)-4-methoxybenzyl)-6-(ethylamino)-8-isopropyl-3H-purinehydrochloride),S,S′-methylene-bis(2-(8-cyclopropyl-3-propyl-6-(4-pyridylmethylamino)-2-thio-3H-purine))tetrahydrochloride, rolipram (2-pyrrolidinone,4-(3-(cyclopentyloxy)-4-methoxyphenyl)-), CP-293121, CP-353164(5-(3-cyclopentyloxy-4-methoxyphenyl)pyridine-2-carboxamide), oxagrelate(6-phthalazinecarboxylic acid,3,4-dihydro-1-(hydroxymethyl)-5,7-dimethyl-4-oxo-, ethyl ester),PD-168787, ibudilast (1-propanone,2-methyl-1-(2-(1-methylethyl)pyrazolo(1,5-a)pyridin-3-yl)-), oxagrelate(6-phthalazinecarboxylic acid,3,4-dihydro-1-(hydroxymethyl)-5,7-dimethyl-4-oxo-, ethyl ester),griseolic acid (alpha-L-talo-oct-4-enofuranuronic acid,1-(6-amino-9H-purin-9-yl)-3,6-anhydro-6-C-carboxy-1,5-dideoxy-),KW-4490, KS-506, T-440, roflumilast (benzamide,3-(cyclopropylmethoxy)-N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-),rolipram, milrinone, triflusinal (benzoic acid,2-(acetyloxy)-4-(trifluoromethyl)-), anagrelide hydrochloride(imidazo[2,1-b]quinazolin-2(3H)-one, 6,7-dichloro-1,5-dihydro-,monohydrochloride), cilostazol(2(1H)-quinolinone,6-(4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy)-3,4-dihydro-),propentofylline (1H-purine-2,6-dione,3,7-dihydro-3-methyl-1-(5-oxohexyl)-7-propyl-), sildenafil citrate(piperazine,1-((3-(4,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo(4,3-d)pyrimidin-5-yl)-4-ethoxyphenyl)sulfonyl)-4-methyl,2-hydroxy-1,2,3-propanetricarboxylate-(1:1)), tadalafil(pyrazino(1′,2′:1,6)pyrido(3,4-b)indole-1,4-dione,6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-,(6R-trans)), vardenafil (piperazine,1-(3-(1,4-dihydro-5-methyl(-4-oxo-7-propylimidazo(5,1-f)(1,2,4)-triazin-2-yl)-4-ethoxyphenyl)sulfonyl)-4-ethyl-),milrinone ((3,4′-bipyridine)-5-carbonitrile,1,6-dihydro-2-methyl-6-oxo-), enoximone (2H-imidazol-2-one,1,3-dihydro-4-methyl-5-(4-(methylthio)benzoyl)-), theophylline(1H-purine-2,6-dione, 3,7-dihydro-1,3-dimethyl-), ibudilast(1-propanone,2-methyl-1-(2-(1-methylethyl)pyrazolo(1,5-a)pyridin-3-yl)-),aminophylline (1H-purine-2,6-dione, 3,7-dihydro-1,3-dimethyl-, compoundwith 1,2-ethanediamine (2:1)-), acebrophylline (7H-purine-7-acetic acid,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-, compd. withtrans-4-(((2-amino-3,5-dibromophenyl)methyl)amino)cyclohexanol (1:1)),plafibride (propanamide,2-(4-chlorophenoxy)-2-methyl-N-(((4-morpholinylmethyl)amino)carbonyl)-),ioprinone hydrochloride (3-pyridinecarbonitrile,1,2-dihydro-5-imidazo[1,2-a]pyridin-6-yl-6-methyl-2-oxo-,monohydrochloride-), fosfosal (benzoic acid, 2-(phosphonooxy)-), aminone((3,4′-bipyridin)-6(1H)-one, 5-amino-, or an analogue or derivativethereof).

Other examples of phosphodiesterase inhibitors include denbufylline(1H-purine-2,6-dione, 1,3-dibutyl-3,7-dihydro-7-(2-oxopropyl)-),propentofylline (1H-purine-2,6-dione,3,7-dihydro-3-methyl-1-(5-oxohexyl)-7-propyl-) and pelrinone(5-pyrimidinecarbonitrile,1,4-dihydro-2-methyl-4-oxo-6-[(3-pyridinylmethyl)amino]-).

Other examples of phosphodiesterase III inhibitors include enoximone(2H-imidazol-2-one, 1,3-dihydro-4-methyl-5-[4-(methylthio)benzoyl]-),and saterinone (3-pyridinecarbonitrile,1,2-dihydro-5-[4-[2-hydroxy-3-[4-(2-methoxyphenyl)-1-piperazinyl]propoxy]phenyl]-6-methyl-2-oxo-).

Other examples of phosphodiesterase IV inhibitors include AWD-12-281,3-auinolinecarboxylic acid,1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-),tadalafil (pyrazino(1′,′:1,6)pyrido(3,4-b)indole-1,4-dione,6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-,(6R-trans)), and filaminast (ethanone,1-[3-(cyclopentyloxy)-4-methoxyphenyl]-, O-(aminocarbonyl)oxime, (1E)-).

Another example of a phosphodiesterase V inhibitor is vardenafil(piperazine,1-(3-(1,4-dihydro-5-methyl(−4-oxo-7-propylimidazo(5,1-f)(1,2,4)-triazin-2-yl)-4-ethoxyphenyl)sulfonyl)-4-ethyl-).

TGF-β Inhibitors

In another embodiment, the pharmacologically active compound is a TGF-βInhibitor (e.g., mannose-6-phosphate, LF-984, tamoxifen (ethanamine,2-(4-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl-, (Z)—), tranilast,or an analogue or derivative thereof).

Thromboxane A2 Antagonists

In another embodiment, the pharmacologically active compound is athromboxane A2 antagonist (e.g., CGS-22652 (3-pyridineheptanoic acid,γ-(4-(((4-chlorophenyl)sulfonyl)amino)butyl)-, (+/−)-), ozagrel(2-propenoic acid, 3-(4-(1H-imidazol-1-ylmethyl)phenyl)-, (E)-),argatroban (2-piperidinecarboxylic acid,1-(5-((aminoiminomethyl)amino)-1-oxo-2-(((1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl)amino)pentyl)-4-methyl-),ramatroban (9H-carbazole-9-propanoic acid,3-(((4-fluorophenyl)sulfonyl)amino)-1,2,3,4-tetrahydro-, (R)—),torasemide (3-pyridinesulfonamide,N-(((1-methylethyl)amino)carbonyl)-4-(3-methylphenyl)amino)-), gammalinoleic acid ((Z,Z,Z)-6,9,12-octadecatrienoic acid), seratrodast(benzeneheptanoic acid,zeta-(2,4,5-trimethyl-3,6-dioxo-1,4-cyclohexadien-1-yl)-, (+/−)-, or ananalogue or derivative thereof).

TNF-α Antagonists and Tace Inhibitors

In another embodiment, the pharmacologically active compound is a TNF-αantagonist or TACE inhibitor (e.g., E-5531(2-deoxy-6-O-(2-deoxy-3-O-(3(R)-(5(Z)-dodecenoyloxy)-decyl)-6-O-methyl-2-(3-oxotetradecanamido)-4-O-phosphono-β-D-glucopyranosyl)-3-O-(3(R)-hydroxydecyl)-2-(3-oxotetradecanamido)-alpha-D-glucopyranose-1-O-phosphate),AZD-4717, glycophosphopeptical, UR-12715 (B=benzoic acid,2-hydroxy-5-((4-(3-(4-(2-methyl-1H-imidazol(4,5-c)pyridin-1-yl)methyl)-1-piperidinyl)-3-oxo-1-phenyl-1-propenyl)phenyl)azo)(Z)), PMS-601, AM-87, xyloadenosine (9H-purin-6-amine,9-β-D-xylofuranosyl-), RDP-58, RDP-59, BB2275, benzydamine, E-3330(undecanoic acid,2-((4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene)-,(E)-),N-(D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl)-L-3-(2′-naphthyl)alanyl-L-alanine,2-aminoethyl amide, CP-564959, MLN-608, SPC-839, ENMD-0997, Sch-23863((2-(10,11-dihydro-5-ethoxy-5H-dibenzo (a,d)cyclohepten-S-yl)-N,N-dimethyl-ethanamine), SH-636, PKF-241-466,PKF-242-484, TNF-484A, cilomilast(cis-4-cyano-4-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexane-1-carboxylicacid), GW-3333, GW-4459, BMS-561392, AM-87, cloricromene (acetic acid,((8-chloro-3-(2-(diethylamino)ethyl)-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy)-,ethyl ester), thalidomide (1H-Isoindole-1,3(2H)-dione,2-(2,6-dioxo-3-piperidinyl)-), vesnarinone (piperazine,1-(3,4-dimethoxybenzoyl)-4-(1,2,3,4-tetrahydro-2-oxo-6-quinolinyl)-),infliximab, lentinan, etanercept (1-235-tumor necrosis factor receptor(human) fusion protein with 236-467-immunoglobulin G1 (humangammal-chain Fc fragment)), diacerein (2-anthracenecarboxylic acid,4,5-bis(acetyloxy)-9,10-dihydro-9,10-dioxo-, or an analogue orderivative thereof).

Tyrosine Kinase Inhibitors

In another embodiment, the pharmacologically active compound is atyrosine kinase inhibitor (e.g., SKI-606, ER-068224, SD-208,N-(6-benzothiazolyl)-4-(2-(1-piperazinyl)pyrid-5-yl)-2-pyrimidineamine,celastrol(24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid,3-hydroxy-9,13-dimethyl-2-oxo-, (9 beta., 13alpha,14β,20 alpha)-),CP-127374 (geldanamycin, 17-demethoxy-17-(2-propenylamino)-), CP-564959,PD-171026, CGP-52411 (1H-Isoindole-1,3(2H)-dione,4,5-bis(phenylamino)-), CGP-53716 (benzamide,N-(4-methyl-3-((4-(3-pyridinyl)-2-pyrimidinyl)amino)phenyl)-), imatinib(4-((methyl-1-piperazinyl)methyl)-N-(4-methyl-3-((4-(3-pyridinyl)-2-pyrimidinyl)amino)-phenyl)benzamidemethanesulfonate), NVP-AAK980-NX, KF-250706(13-chloro,5(R),6(S)-epoxy-14,16-dihydroxy-11-(hydroyimino)-3(R)-methyl-3,4,5,6,11,12-hexahydro-1H-2-benzoxacyclotetradecin-1-one),5-(3-(3-methoxy-4-(2-((E)-2-phenylethenyl)-4-oxazolylmethoxy)phenyl)propyl)-3-(2-((E)-2-phenylethenyl)-4-oxazolylmethyl)-2,4-oxazolidinedione,genistein, NV-06, or an analogue or derivative thereof).

Vitronectin Inhibitors

In another embodiment, the pharmacologically active compound is avitronectin inhibitor (e.g.,O-(9,10-dimethoxy-1,2,3,4,5,6-hexahydro-4-((1,4,5,6-tetrahydro-2-pyrimidinyl)hydrazono)-8-benz(e)azulenyl)-N-((phenylmethoxy)carbonyl)-DL-homoserine2,3-dihydroxypropyl ester, (2S)-benzoylcarbonylamino-3-(2-((4S)-(3-(4,5-dihydro-1H-imidazol-2-ylamino)-propyl)-2,5-dioxo-imidazolidin-1-yl)-acetylamino)-propionate,Sch-221153, S-836, SC-68448(β-((2-2-(((3-((aminoiminomethyl)amino)-phenyl)carbonyl)amino)acetyl)amino)-3,5-dichlorobenzenepropanoicacid), SD-7784, S-247, or an analogue or derivative thereof).

Fibroblast Growth Factor Inhibitors

In another embodiment, the pharmacologically active compound is afibroblast growth factor inhibitor (e.g., CT-052923(((2H-benzo(d)1,3-dioxalan-5-methyl)amino)(4-(6,7-dimethoxyquinazolin-4-yl)piperazinyl)methane-1-thione),or an analogue or derivative thereof).

Protein Kinase Inhibitors

In another embodiment, the pharmacologically active compound is aprotein kinase inhibitor (e.g., KP-0201448, NPC15437 (hexanamide,2,6-diamino-N-((1-(1-oxotridecyl)-2-piperidinyl)methyl)-), fasudil(1H-1,4-diazepine, hexahydro-1-(5-isoquinolinylsulfonyl)-), midostaurin(benzamide,N-(2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9H-diindolo(1,2,3-gh:3′,2′,1′-lm)pyrrolo(3,4-j)(1,7)benzodiazonin-11-yl)-N-methyl-,(9Alpha,10β,11β,13Alpha)-),fasudil (1H-1,4-diazepine,hexahydro-1-(5-isoquinolinylsulfonyl)-, dexniguldipine(3,5-pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-,3-(4,4-diphenyl-1-piperidinyl)propyl methyl ester, monohydrochloride,(R)—), LY-317615 (1H-pyrole-2,5-dione,3-(1-methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-,monohydrochloride), perifosine (piperidinium,4-[[hydroxy(octadecyloxy)phosphinyl]oxy]-1,1-dimethyl-, inner salt),LY-333531 (9H,18H-5,21:12,17-dimethenodibenzo(e,k)pyrrolo(3,4-h)(1,4,13)oxadiazacyclohexadecine-18,20(19H)-dione,9-((dimethylamino)methyl)-6,7,10,11-tetrahydro-,(S)—), Kynac; SPC-100270 (1,3-octadecanediol, 2-amino-, [S—(R*,R*)]-),Kynacyte, or an analogue or derivative thereof).

PDGF Receptor Kinase Inhibitors

In another embodiment, the pharmacologically active compound is a PDGFreceptor kinase inhibitor (e.g., RPR-127963E, or an analogue orderivative thereof).

Endothelial Growth Factor Receptor Kinase Inhibitors

In another embodiment, the pharmacologically active compound is anendothelial growth factor receptor kinase inhibitor (e.g., CEP-7055,SU-0879((E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(aminothiocarbonyl)acrylonitrile),BIBF-1000, AG-013736 (CP-868596), AMG-706, AVE-0005, NM-3(3-(2-methylcarboxymethyl)-6-methoxy-8-hydroxy-isocoumarin),Bay-43-9006, SU-011248, or an analogue or derivative thereof).

Retinoic Acid Receptor Antagonists

In another embodiment, the pharmacologically active compound is aretinoic acid receptor antagonist (e.g., etarotene (Ro-15-1570)(naphthalene,6-(2-(4-(ethylsulfonyl)phenyl)-1-methylethenyl)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-,(E)-),(2E,4E)-3-methyl-5-(2-((E)-2-(2,6,6-trimethyl-1-cyclohexen-1-yl)ethenyl)-1-cyclohexen-1-yl)-2,4-pentadienoicacid, tocoretinate (retinoic acid,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-ylester, (2R*(4R*,8R*))-(±)-), aliretinoin (retinoic acid, cis-9,trans-13-), bexarotene (benzoic acid,4-(1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl)-),tocoretinate (retinoic acid,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-ylester, [2R*(4R*,8R*)]-(±)-, or an analogue or derivative thereof).

Platelet Derived Growth Factor Receptor Kinase Inhibitors

In another embodiment, the pharmacologically active compound is aplatelet derived growth factor receptor kinase inhibitor (e.g.,leflunomide (4-isoxazolecarboxamide,5-methyl-N-(4-(trifluoromethyl)phenyl)-, or an analogue or derivativethereof).

Fibrinogen Antagonists

In another embodiment, the pharmacologically active compound is afibrinogen antagonist (e.g., picotamide (1,3-benzenedicarboxamide,4-methoxy-N,N′-bis(3-pyridinylmethyl)-, or an analogue or derivativethereof).

Antimycotic Agents

In another embodiment, the pharmacologically active compound is anantimycotic agent (e.g., miconazole, sulconizole, parthenolide,rosconitine, nystatin, isoconazole, fluconazole, ketoconasole,imidazole, itraconazole, terpinafine, elonazole, bifonazole,clotrimazole, conazole, terconazole (piperazine,1-(4-((2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(1-methylethyl)-,cis-), isoconazole(1-(2-(2-6-dichlorobenzyloxy)-2-(2-,4-dichlorophenyl)ethyl)),griseofulvin (spiro(benzofuran-2(3H), 1′-(2)cyclohexane)-3,4′-dione,7-chloro-2′,4,6-trimeth-oxy-6′ methyl-, (1′S-trans)-), bifonazole(1H-imidazole, 1-((1,1′-biphenyl)-4-ylphenylmethyl)-), econazole nitrate(1-(24(4-chlorophenyl)methoxy)-2-(2,4-dichlorophenyl)ethyl)-1H-imidazolenitrate), croconazole (1H-imidazole,1-(1-(2-((3-chlorophenyl)methoxy)phenyl)ethenyl)-), sertaconazole(1H-Imidazole,1-(2-((7-chlorobenzo(b)thien-3-yl)methoxy)-2-(2,4-dichlorophenyl)ethyl)-),omoconazole (1H-imidazole,1-(2-(2-(4-chlorophenoxy)ethoxy)-2-(2,4-dichlorophenyl)-1-methylethenyl)-,(Z)—), flutrimazole (1H-imidazole,1-((2-fluorophenyl)(4-fluorophenyl)phenylmethyl)-), fluconazole(1H-1,2,4-triazole-1-ethanol,alpha-(2,4-difluorophenyl)-alpha-(1H-1,2,4-triazol-1-ylmethyl)-),neticonazole (1H-Imidazole,1-(2-(methylthio)-1-(2-(pentyloxy)phenyl)ethenyl)-, monohydrochloride,(E)-), butoconazole (1H-imidazole,1-(4-(4-chlorophenyl)-2-(2,6-dichlorophenyl)thio)butyl)-, (+/−)-),clotrimazole (1((2-chlorophenyl)diphenylmethyl)-1H-imidazole, or ananalogue or derivative thereof).

Bisphosphonates

In another embodiment, the pharmacologically active compound is abisphosphonate (e.g., clodronate, alendronate, pamidronate, zoledronate,or an analogue or derivative thereof).

Phospholipase A1 Inhibitors

In another embodiment, the pharmacologically active compound is aphospholipase A1 inhibitor (e.g., ioteprednol etabonate(androsta-1,4-diene-17-carboxylic acid,17-((ethoxycarbonyl)oxy)-11-hydroxy-3-oxo-, chloromethyl ester, (11β,17alpha)-, or an analogue or derivative thereof).

Histamine H1/H2/H3 Receptor Antagonists

In another embodiment, the pharmacologically active compound is ahistamine H1, H2, or H3 receptor antagonist (e.g., ranitidine(1,1-ethenediamine,N-(2-(((5-((dimethylamino)methyl)-2-furanyl)methyl)thio)ethyl)-N′-methyl-2-nitro-),niperotidine(N-(24(5-((dimethylamino)methyl)furfuryl)thio)ethyl)-2-nitro-N′-piperonyl-1,1-ethenediamine),famotidine (propanimidamide,3-(((2-((aminoiminomethyl)amino)-4-thiazolyl)methyl)thio)-N-(aminosulfonyl)-),roxitadine acetate HCl (acetamide,2-(acetyloxy)-N-(3-(3-(1-piperidinylmethyl)phenoxy)propyl)-,monohydrochloride), lafutidine (acetamide,2-((2-furanylmethyl)sulfinyl)-N-(4-((4-(1-piperidinylmethyl)-2-pyridinyl)oxy)-2-butenyl)-,(Z)—), nizatadine (1,1-ethenediamine,N-(2-(((2-((dimethylamino)methyl)-4-thiazolyl)methyl)thio)ethyl)-N′-methyl-2-nitro-),ebrotidine (benzenesulfonamide,N-(((2-(((2-((aminoiminomethyl)amino)-4-thiazoly)methyl)thio)ethyl)amino)methylene)-4-bromo-),rupatadine (5H-benzo (5,6)cyclohepta(1,2-b)pyridine,8-chloro-6,11-dihydro-11-(1-((5-methyl-3-pyridinyl)methyl)-4-piperidinylidene)-,trihydrochloride-), fexofenadine HCl (benzeneacetic acid,4-(1-hydroxy-4-(4(hydroxydiphenylmethyl)-1-piperidinyl)butyl)-alpha,alpha-dimethyl-, hydrochloride, or an analogue or derivative thereof).

Macrolide Antibiotics

In another embodiment, the pharmacologically active compound is amacrolide antibiotic (e.g., dirithromycin (erythromycin,9-deoxo-11-deoxy-9,11-(imino(2-(2-methoxyethoxy)ethylidene)oxy)-,(9S(R))-), flurithromycin ethylsuccinate (erythromycin,8-fluoro-mono(ethyl butanedioate) (ester)-), erythromycin stinoprate(erythromycin, 2′-propanoate, compound with N-acetyl-L-cysteine (1:1)),clarithromycin (erythromycin, 6-O-methyl-), azithromycin(9-deoxo-9a-aza-9a-methyl-9a-homoerythromycin-A), telithromycin(3-de((2,6-dideoxy-3-C-methyl-3-O-methyl-alpha-L-ribo-hexopyranosyl)oxy)-11,12-dideoxy-6-O-methyl-3-oxo-12,11-(oxycarbonyl((4-(4-(3-pyridinyl)-1H-imidazol-1-yl)butyl)imino))-),roxithromycin (erythromycin, 9-(O-((2-methoxyethoxy)methyl)oxime)),rokitamycin (leucomycin V, 4B-butanoate 3B-propanoate), RV-11(erythromycin monopropionate mercaptosuccinate), midecamycin acetate(leucomycin V, 3B,9-diacetate 3,4B-dipropanoate), midecamycin(leucomycin V, 3,4B-dipropanoate), josamycin (leucomycin V, 3-acetate4B-(3-methylbutanoate), or an analogue or derivative thereof).

GPIIb IIIa Receptor Antagonists

In another embodiment, the pharmacologically active compound is a GPIIbIIIa receptor antagonist (e.g., tirofiban hydrochloride (L-tyrosine,N-(butylsulfonyl)-O-(4-(4-piperidinyl)butyl)-, monohydrochloride-),eptifibatide (L-cysteinamide,N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl)-L-lysylglycyl-L-alpha-aspartyl-L-tryptophyl-L-prolyl-,cyclic(1->6)-disulfide), xemilofiban hydrochloride, or an analogue orderivative thereof).

Endothelin Receptor Antagonists

In another embodiment, the pharmacologically active compound is anendothelin receptor antagonist (e.g., bosentan (benzenesulfonamide,4-(1,1-dimethylethyl)-N-(6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)(2,2′-bipyrimidin)-4-yl)-,or an analogue or derivative thereof).

Peroxisome Proliferator-Activated Receptor Agonists

In another embodiment, the pharmacologically active compound is aperoxisome proliferator-activated receptor agonist (e.g., gemfibrozil(pentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl-), fenofibrate(propanoic acid, 2-(4-(4-chlorobenzoyl)phenoxy)-2-methyl-, 1-methylethylester), ciprofibrate (propanoic acid,2-(4-(2,2-dichlorocyclopropyl)phenoxy)-2-methyl-), rosiglitazone maleate(2,4-thiazolidinedione,5-((4-(2-(methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-,(Z)-2-butenedioate (1:1)), pioglitazone hydrochloride(2,4-thiazolidinedione,5-((4-(2-(5-ethyl-2-pyridinyl)ethoxy)phenyl)methyl)-, monohydrochloride(+/−)-), etofylline clofibrate (propanoic acid,2-(4-chlorophenoxy)-2-methyl-,2-(1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purin-7-yl)ethyl ester),etofibrate (3-pyridinecarboxylic acid,2-(2-(4-chlorophenoxy)-2-methyl-1-oxopropoxy)ethyl ester), clinofibrate(butanoic acid,2,2′-(cyclohexylidenebis(4,1-phenyleneoxy))bis(2-methyl-)), bezafibrate(propanoic acid,2-(4-(2-((4-chlorobenzoyl)amino)ethyl)phenoxy)-2-methyl-), binifibrate(3-pyridinecarboxylic acid,2-(2-(4-chlorophenoxy)-2-methyl-1-oxopropoxy)-1,3-propanediyl ester), oran analogue or derivative thereof).

In one aspect, the pharmacologically active compound is a peroxisomeproliferator-activated receptor alpha agonist, such as GW-590735,GSK-677954, GSK501516, pioglitazone hydrochloride(2,4-thiazolidinedione,5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-, monohydrochloride(+/−)-, or an analogue or derivative thereof).

Estrogen Receptor Agents

In another embodiment, the pharmacologically active compound is anestrogen receptor agent (e.g., estradiol, 17-β-estradiol, or an analogueor derivative thereof).

Somatostatin Analogues

In another embodiment, the pharmacologically active compound is asomatostatin analogue (e.g., angiopeptin, or an analogue or derivativethereof).

Neurokinin 1 Antagonists

In another embodiment, the pharmacologically active compound is aneurokinin 1 antagonist (e.g., GW-597599, lanepitant((1,4′-bipiperidine)-1′-acetamide,N-(2-(acetyl((2-methoxyphenyl)methyl)amino)-1-(1H-indol-3-ylmethyl)ethyl)-(R)—),nolpitantium chloride (1-azoniabicyclo[2.2.2]octane,1-[2-[3-(3,4-dichlorophenyl)-1-[[3-(1-methylethoxy)phenyl]acetyl]-3-piperidinyl]ethyl]-4-phenyl-,chloride, (S)—), or saredutant (benzamide,N-[4-[4-(acetylamino)-4-phenyl-1-piperidinyl]-2-(3,4-dichlorophenyl)butyl]-N-methyl-,(S)—), or vofopitant (3-piperidinamine,N—[[2-methoxy-5-[5-(trifluoromethyl)-1H-tetrazol-1-yl]phenyl]methyl]-2-phenyl-,(2S,3S)—, or an analogue or derivative thereof).

Neurokinin 3 Antagonist

In another embodiment, the pharmacologically active compound is aneurokinin 3 antagonist (e.g., talnetant (4-quinolinecarboxamide,3-hydroxy-2-phenyl-N-[(1S)-1-phenylpropyl]-, or an analogue orderivative thereof).

Neurokinin Antagonist

In another embodiment, the pharmacologically active compound is aneurokinin antagonist (e.g., GSK-679769, GSK-823296, SR-489686(benzamide,N-[4-[4-(acetylamino)-4-phenyl-1-piperidinyl]-2-(3,4-dichlorophenyl)butyl]-N-methyl-,(S)—), SB-223412; SB-235375 (4-quinolinecarboxamide,3-hydroxy-2-phenyl-N-[(1S)-1-phenylpropyl]-), UK-226471, or an analogueor derivative thereof).

VLA-4 Antagonist

In another embodiment, the pharmacologically active compound is a VLA-4antagonist (e.g., GSK683699, or an analogue or derivative thereof).

Osteoclast Inhibitor

In another embodiment, the pharmacologically active compound is aosteoclast inhibitor (e.g., ibandronic acid (phosphonic acid,[1-hydroxy-3-(methylpentylamino)propylidene]bis-), alendronate sodium,or an analogue or derivative thereof).

DNA Topoisomerase ATP Hydrolysing Inhibitor

In another embodiment, the pharmacologically active compound is a DNAtopoisomerase ATP hydrolysing inhibitor (e.g., enoxacin(1,8-naphthyridine-3-carboxylic acid,1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-), levofloxacin(7H-Pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid,9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-, (S)—),ofloxacin (7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid,9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-,(+/−)-), pefloxacin (3-quinolinecarboxylic acid,1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-),pipemidic acid (pyrido[2,3-d]pyrimidine-6-carboxylic acid,8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)-), pirarubicin(5,12-naphthacenedione,10-[[3-amino-2,3,6-trideoxy-4-O-(tetrahydro-2H-pyran-2-yl)-alpha-L-lyxo-hexopyranosyl]oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-,[8S-[8 alpha,10 alpha(S*)]]-), sparfloxacin (3-quinolinecarboxylic acid,5-amino-1-cyclopropyl-7-(3,5-dimethyl-1-piperazinyl)-6,8-difluoro-1,4-dihydro-4-oxo-,cis-), AVE-6971, cinoxacin ([1,3]dioxolo[4,5-g]cinnoline-3-carboxylicacid, 1-ethyl-1,4-dihydro-4-oxo-), or an analogue or derivativethereof).

Angiotensin I Converting Enzyme Inhibitor

In another embodiment, the pharmacologically active compound is anangiotensin I converting enzyme inhibitor (e.g., ramipril(cyclopenta[b]pyrrole-2-carboxylic acid,1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]octahydro-,[2S-[1[R*(R*)],2 alpha, 3aβ, 6aβ]]-), trandolapril(1H-indole-2-carboxylic acid,1-[2-[(1-carboxy-3-phenylpropyl)amino]-1-oxopropyl]octahydro-,[2S-[1[R*(R*)],2 alpha,3a alpha,7aβ]]-), fasidotril (L-alanine,N-[(2S)-3-(acetylthio)-2-(1,3-benzodioxol-5-ylmethyl)-1-oxopropyl]-,phenylmethyl ester), cilazapril(6H-pyridazino[1,2-a][1,2]diazepine-1-carboxylic acid,9-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]octahydro-10-oxo-, [1S-[1alpha, 9 alpha(R*)]]-), ramipril (cyclopenta[b]pyrrole-2-carboxylicacid,1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]octahydro-,[2S-[1[R*(R*)], 2 alpha,3aβ,6aβ]]-, or an analogue or derivativethereof).

Angiotensin II Antagonist

In another embodiment, the pharmacologically active compound is anangiotensin II antagonist (e.g., HR-720 (1H-imidazole-5-carboxylic acid,2-butyl-4-(methylthio)-1-[[2′-[[[(propylamino)carbonyl]amino]sulfonyl][1,1′-biphenyl]-4-yl]methyl]-,dipotassium salt, or an analogue or derivative thereof).

Enkephalinase Inhibitor

In another embodiment, the pharmacologically active compound is anenkephalinase inhibitor (e.g., Aventis 100240(pyrido[2,1-a][2]benzazepine-4-carboxylic acid,7-[[2-(acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8,12b-octahydro-6-oxo-,[4S-[4 alpha, 7 alpha(R*),12b13]]-), AVE-7688, or an analogue orderivative thereof).

Peroxisome Proliferator-Activated Receptor γ Agonist Insulin Sensitizer

In another embodiment, the pharmacologically active compound isperoxisome proliferator-activated receptor γ agonist insulin sensitizer(e.g., rosiglitazone maleate (2,4-thiazolidinedione,5-((4-(2-(methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-,(Z)-2-butenedioate (1:1), farglitazar (GI-262570, GW-2570, GW-3995,GW-5393, GW-9765), LY-929, LY-519818, LY-674, or LSN-862), or ananalogue or derivative thereof).

Protein Kinase C Inhibitor

In another embodiment, the pharmacologically active compound is aprotein kinase C inhibitor, such as ruboxistaurin mesylate (9H,18H-5,21:12,17-dimethenodibenzo(e,k)pyrrolo(3,4-h)(1,4,13)oxadiazacyclohexadecine-18,20(19H)-dione,9-((dimethylamino)methyl)-6,7,10,11-tetrahydro-,(S)—), safingol (1,3-octadecanediol, 2-amino-, [S—(R*,R*)]-), orenzastaurin hydrochloride (1H-pyrole-2,5-dione,3-(1-methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-,monohydrochloride), or an analogue or derivative thereof.

Rock (Rho-Assoclated Kinase) Inhibitors

In another embodiment, the pharmacologically active compound is a ROCK(rho-associated kinase) inhibitor, such as Y-27632, HA-1077, H-1152 and4-1-(aminoalkyl)-N-(4-pyridyl) cyclohexanecarboxamide or an analogue orderivative thereof.

CXCR3Inhibitors

In another embodiment, the pharmacologically active compound is a CXCR3inhibitor such as T-487, T0906487 or analogue or derivative thereof.

ITK Inhibitors

In another embodiment, the pharmacologically active compound is an Itkinhibitor such as BMS-509744 or an analogue or derivative thereof.

Cytosolic Phospholipase A₂-Alpha Inhibitors

In another embodiment, the pharmacologically active compound is acytosolic phospholipase A₂-alpha inhibitor such as efipladib (PLA-902)or analogue or derivative thereof.

PPAR Agonist

In another embodiment, the pharmacologically active compound is a PPARAgonist (e.g., Metabolex ((−)-benzeneacetic acid,4-chloro-alpha-[3-(trifluoromethyl)-phenoxy]-, 2-(acetylamino)ethylester), balaglitazone(5-(4-(3-methyl-4-oxo-3,4-dihydro-quinazolin-2-yl-methoxy)-benzyl)-thiazolidine-2,4-dione),ciglitazone (2,4-thiazolidinedione,5-[[4-[(1-methylcyclohexyl)methoxy]phenyl]methyl]-), DRF-10945,farglitazar, GSK-677954, GW-409544, GW-501516, GW-590735, GW-590735,K-111, KRP-101, LSN-862, LY-519818, LY-674, LY-929, muraglitazar;BMS-298585 (Glycine,N-[(4-methoxyphenoxy)carbonyl]-N-[[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]methyl]-),netoglitazone; isaglitazone (2,4-thiazolidinedione,5-[[6-[(2-fluorophenyl)methoxy]-2-naphthalenyl]methyl]-, Actos AD-4833;U-72107A (2,4-thiazolidinedione,5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-, monohydrochloride(+/−)-), JTT-501; PNU-182716 (3,5-Isoxazolidinedione,4-[[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]methyl]-), AVANDIA(from SB Pharmco Puerto Rico, Inc. (Puerto Rico);BRL-48482;BRL-49653;BRL-49653c; NYRACTA and Venvia (both from(SmithKline Beecham (United Kingdom)); tesaglitazar((2S)-2-ethoxy-3-[4-[2-[4-[(methylsulfonyl)oxy]phenyl]ethoxy]phenyl]propanoicacid), troglitazone (2,4-Thiazolidinedione,5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-),and analogues and derivatives thereof).

Immunosuppressants

In another embodiment, the pharmacologically active compound is animmunosuppressant (e.g., batebulast (cyclohexanecarboxylic acid,4-[[(aminoiminomethyl)amino]methyl]-, 4-(1,1-dimethylethyl)phenyl ester,trans-), cyclomunine, exalamide (benzamide, 2-(hexyloxy)-), LYN-001,CCl-779 (rapamycin 42-(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate)),1726; 1726-D; AVE-1726, or an analogue or derivative thereof).

ERB Inhibitor

In another embodiment, the pharmacologically active compound is an Erbinhibitor (e.g., canertinib dihydrochloride(N-[4-(3-(chloro-4-fluoro-phenylamino)-7-(3-morpholin-4-yl-propoxy)-quinazolin-6-yl]-acrylamidedihydrochloride), CP-724714, or an analogue or derivative thereof).

Apoptosis Agonist

In another embodiment, the pharmacologically active compound is anapoptosis agonist (e.g., CEFLATONIN® (CGX-635) (from ChemgenexTherapeutics, Inc., Menlo Park, Calif.), CHML, LBH-589, metoclopramide(benzamide, 4-amino-5-chloro-N-[2-(diethylamino)ethyl]-2-methoxy-),patupilone (4,17-dioxabicyclo(14.1.0)heptadecane-5,9-dione,7,11-dihydroxy-8,8,10,12,16-pentamethyl-3-(1-methyl-2-(2-methyl-4-thiazolyl)ethenyl,(1R,3S,7S,10R,11S,12S,16R)), AN-9; pivanex (butanoic acid,(2,2-dimethyl-1-oxopropoxy)methyl ester), SL-100; SL-102; SL-11093;SL-11098; SL-11099; SL-93; SL-98; SL-99, or an analogue or derivativethereof).

Lipocortin Agonist

In another embodiment, the pharmacologically active compound is anlipocortin agonist (e.g., CGP-13774(9Alpha-chloro-6Alpha-fluoro-11β,17alpha-dihydroxy-16Alpha-methyl-3-oxo-1,4-androstadiene-17β-carboxylicacid-methylester-17-propionate), or analogue or derivative thereof).

VCAM-1 Antagonist

In another embodiment, the pharmacologically active compound is a VCAM-1antagonist (e.g., DW-908e, or an analogue or derivative thereof).

Collagen Antagonist

In another embodiment, the pharmacologically active compound is acollagen antagonist (e.g., E-5050 (Benzenepropanamide,4-(2,6-dimethylheptyl)-N-(2-hydroxyethyl)-β-methyl-), lufironil(2,4-Pyridinedicarboxamide, N,N′-bis(2-methoxyethyl)-), or an analogueor derivative thereof).

Alpha 2 Integrin Antagonist

In another embodiment, the pharmacologically active compound is an alpha2 integrin antagonist (e.g., E-7820, or an analogue or derivativethereof).

TNF-α Inhibitor

In another embodiment, the pharmacologically active compound is a TNF-αinhibitor (e.g., ethyl pyruvate, Genz-29155, lentinan (Ajinomoto Co.,Inc. (Japan)), linomide (3-quinolinecarboxamide,1,2-dihydro-4-hydroxy-N,1-dimethyl-2-oxo-N-phenyl-), UR-1505, or ananalogue or derivative thereof).

Nitric Oxide Inhibitor

In another embodiment, the pharmacologically active compound is a nitricoxide (NO) inhibitor (e.g., guanidioethyldisulfide, or an analogue orderivative thereof).

Cathepsin Inhibitor

In another embodiment, the pharmacologically active compound is acathepsin inhibitor (e.g., SB-462795 or an analogue or derivativethereof).

C. Delivery of Cells and Genes

The multifunctional compounds of the invention can also be used todeliver various types of living cells or genes to a desired site ofadministration in order to form new tissue. The term “genes” as usedherein is intended to encompass genetic material from natural sources,synthetic nucleic acids, DNA, antisense-DNA and RNA. Thus, this aspectof the invention is a method for delivering living cells or genes, wherethe composition also includes the cells of genes to be delivered, andsteps (a) and (b) are as described for the method of sealing tissue.Step (c) would involve allowing a three-dimensional matrix to form anddelivering the cells or genes.

When used to deliver cells, for example, mesenchymal stem cells can bedelivered to produce cells of the same type as the tissue into whichthey are delivered. Mesenchymal stem cells are not differentiated andtherefore can differentiate to form various types of new cells due tothe presence of an active agent or the effects (chemical, physical,etc.) of the local tissue environment. Examples of mesenchymal stemcells include osteoblasts, chondrocytes, and fibroblasts. Osteoblastscan be delivered to the site of a bone defect to produce new bone;chondrocytes can be delivered to the site of a cartilage defect toproduce new cartilage; fibroblasts can be delivered to produce collagenwherever new connective tissue is needed; neurectodermal cells can bedelivered to form new nerve tissue; epithelial cells can be delivered toform new epithelial tissues, such as liver, pancreas, etc.

The cells or genes may be either allogeneic or xenogeneic in origin. Forexample, the multifunctional compounds can be used to deliver cells orgenes from other species that have been genetically modified. Becausethe compounds of the invention are not easily degraded in vivo, cellsand genes entrapped within the three-dimensional matrix will be isolatedfrom the patient's own cells and, as such, will not provoke an immuneresponse in the patient. In order to entrap the cells or genes withinthis matrix, the cells or genes are pre-mixed with the multifunctionalcompounds in an initial environment. Upon exposure to the modifiedenvironment, a three-dimensional matrix is formed, thereby entrappingthe cells or genes within the matrix.

As discussed above for biologically active agents, when used to delivercells or genes, the multifunctional compound may also containbiodegradable groups to aid in controlled release of the cells or genesat the intended site of delivery.

D. Bioadhesives

As used herein, the terms “bioadhesive,” “biological adhesive,” and“surgical adhesive” are used interchangeably to refer to biocompatiblecompositions capable of effecting temporary or permanent attachmentbetween the surfaces of two native tissues, or between a native tissuesurface and either a non-native tissue surface or a surface of asynthetic implant.

In a general method for effecting the attachment of a first surface to asecond surface, the multifunctional compound is applied to a firstsurface, which is then contacted with a second surface to effectadhesion therebetween. Preferably, the multifunctional compound isexposed to the modified environment to initiate reaction among thereactive groups and then delivered to the first surface beforesubstantial reaction has occurred. The first surface is then contactedwith the second surface, preferably immediately, to effect adhesion.Thus, another embodiment of the invention is a method of bioadhesionbetween two surfaces, where steps (a) and (b) are as described for themethod of sealing tissue, and step (c) involves allowing athree-dimensional matrix to form and adhere the surfaces.

The two surfaces may be held together manually, or using otherappropriate means, while the reaction is proceeding to completion.Reaction is typically sufficiently complete for adhesion to occur withinabout 5 to 60 minutes after exposure of the multifunctional compound tothe modified environment. However, the time required for completereaction to occur is dependent on a number of factors, including thetype and molecular weight of each reactive component, the degree offunctionalization, and the concentration of the multifunctional compound(i.e., higher concentrations result in faster reaction times).

At least one of the first and second surfaces is preferably a nativetissue surface. As used herein, the term “native tissue” refers tobiological tissues that are native to the body of the patient beingtreated. As used herein, the term “native tissue” is intended to includebiological tissues that have been elevated or removed from one part ofthe body of a patient for implantation to another part of the body ofthe same patient (such as bone autografts, skin flap autografts, etc.).For example, the multifunctional compounds of the invention can be usedto adhere a piece of skin from one part of a patient's body to anotherpart of the body, as in the case of a burn victim.

The other surface may be a native tissue surface, a non-native tissuesurface, or a surface of a synthetic implant. As used herein, the term“non-native tissue” refers to biological tissues that have been removedfrom the body of a donor patient (who may be of the same species or of adifferent species than the recipient patient) for implantation into thebody of a recipient patient (e.g., tissue and organ transplants). Forexample, the multifunctional compounds of the invention can be used toadhere a donor cornea to the eye of a recipient patient. As used herein,the term “synthetic implant” refers to any biocompatible materialintended for implantation into the body of a patient not encompassed bythe above definitions for native tissue and non-native tissue. Syntheticimplants include, for example, artificial blood vessels, heart valves,artificial organs, bone prostheses, implantable lenticules, vasculargrafts, stents, and stent/graft combinations, etc.

E. Ophthalmic Applications

Because of their optical clarity, the multifunctional compounds of theinvention are particularly well suited for use in ophthalmicapplications. See for example, Margalit et al. “Bioadhesives forIntraocular Use,” Retina 20:469-477 (2000). For example, a syntheticlenticule for correction of vision can be attached to the Bowman's layerof the cornea of a patient's eye using the methods of the presentinvention. In a manner similar to that described in U.S. Pat. No.5,565,519 to Rhee et al., the multifunctional compounds of the inventioncan be molded into a desired lenticular shape, either during or afterformation of the three-dimensional matrix. The resulting collagenlenticule can then be attached to the Bowman's layer of ade-epithelialized cornea of a patient's eye using the methods of thepresent invention. By applying the multifunctional compounds to theanterior surface of the cornea, then contacting the anterior surface ofthe cornea with the posterior surface of the lenticule beforesubstantial reaction has occurred, reactive groups (e.g., electrophilicgroups) on the compounds will also covalently bind to collagen moleculesin both the corneal tissue and the lenticule to firmly anchor thelenticule in place. Alternatively, the multifunctional compounds can beapplied first to the posterior surface of the lenticule, which is thencontacted with the anterior surface of the cornea.

Thus, another embodiment of the invention is a method of ophthalmicrepair of the cornea, where steps (a) and (b) are as described for themethod of sealing tissue, and step (c) involves allowing athree-dimensional matrix to form and adhering a lenticule to the cornea.

The compositions of the present invention are also suitable for use invitreous replacement. In addition, the compositions of the presentinvention may be used for the delivery of active agents.

F. Tissue Augmentation

The multifunctional compounds of the invention can also be used foraugmentation of soft or hard tissue within the body of a mammaliansubject. As such, they may be better than currently marketedcollagen-based materials for soft tissue augmentation, because they areless immunogenic and more persistent. Examples of soft tissueaugmentation applications include sphincter (e.g., urinary, anal,esophageal) augmentation and the treatment of rhytids and scars.Examples of hard tissue augmentation applications include the repairand/or replacement of bone and/or cartilaginous tissue. Thus, anotherembodiment of the invention is a method of tissue augmentation for apre-selected site, where steps (a) and (b) are as described for themethod of sealing tissue, and step (c) involves allowing athree-dimensional matrix to form at the pre-selected site.

The multifunctional compounds are particularly suited for use as areplacement material for synovial fluid in osteoarthritic joints,serving to reduce joint pain and improve joint function by restoring asoft hydrogel network in the joint. The compounds can also be used as areplacement material for the nucleus pulposus of a damagedintervertebral disk. The nucleus pulposus of the damaged disk is firstremoved, and the multifunctional compounds is then injected or otherwiseintroduced into the center of the disk. The compounds may either beexposed to the modified environment prior to introduction into the disk,or allowed to inter-react in situ.

In a general method for effecting augmentation of tissue within the bodyof a mammalian subject, the multifunctional compounds are injectedsimultaneously with exposure to the modified environment, to a tissuesite in need of augmentation through a small-gauge (e.g., 25-32 gauge)needle. Once inside the patient's body, the reactive groups on themultifunctional compounds inter-react with each other to form athree-dimensional matrix in situ. In addition, in some embodiments ofthe invention, the reactive groups on the multifunctional compounds canreact with body tissue to further enhance tissue augmentation. Forexample, when some of the reactive groups are electrophilic groups, suchgroups may also react with primary amino groups on lysine residues ofcollagen molecules within the patient's own tissue, providing for“biological anchoring” of the compositions with the host tissue.

G. Adhesion Prevention

Another use of the multifunctional compounds of the invention is to coattissues in order to prevent the formation of adhesions following surgeryor injury to internal tissues or organs.

Surgical adhesions are abnormal, fibrous bands of scar tissue that canform inside the body as a result of the healing process that follows anyopen or minimally invasive surgical procedure including abdominal,gynecologic, cardiothoracic, spinal, plastic, vascular, ENT,ophthalmologic, urologic, neuro, or orthopedic surgery. Surgicaladhesions are typically connective tissue structures that form betweenadjacent injured areas within the body. Briefly, localized areas ofinjury trigger an inflammatory and healing response that culminates inhealing and scar tissue formation. If scarring results in the formationof fibrous tissue bands or adherence of adjacent anatomical structures(that should be separate), surgical adhesion formation is said to haveoccurred. Adhesions can range from flimsy, easily separable structuresto dense, tenacious fibrous structures that can only be separated bysurgical dissection. While many adhesions are benign, some can causesignificant clinical problems and are a leading cause of repeat surgicalintervention.

Since interventions involve a certain degree of trauma to the operativetissues, virtually any procedure (no matter how well executed) has thepotential to result in the formation of clinically significant adhesionformation. Adhesions can be triggered by surgical trauma such ascutting, manipulation, retraction or suturing, as well as frominflammation, infection (e.g., fungal or mycobacterium), bleeding or thepresence of a foreign body. Surgical trauma may also result from tissuedrying, ischemia, or thermal injury. Due to the diverse etiology ofsurgical adhesions, the potential for formation exists regardless ofwhether the surgery is done in a so-called minimally invasive fashion(e.g., catheter-based therapies, laparoscopy) or in a standard opentechnique involving one or more relatively large incisions. Although apotential complication of any surgical intervention, surgical adhesionsare particularly problematic in GI surgery (causing bowel obstruction),gynecological surgery (causing pain and/or infertility), tendon repairs(causing shortening and flexion deformities), joint capsule procedures(causing capsular contractures), and nerve and muscle repair procedures(causing diminished or lost function).

The placement of medical devices and implants also increases the riskthat surgical adhesions will occur. In addition to the above mechanisms,an implanted device can trigger a “foreign body” response where theimmune system recognizes the implant as foreign and triggers aninflammatory reaction that ultimately leads to scar tissue formation. Aspecific form of foreign body reaction in response to medical deviceplacement is complete enclosure (“walling off”) of the implant in acapsule of scar tissue (encapsulation). Fibrous encapsulation ofimplanted devices and implants can complicate any procedure, but breastaugmentation and reconstruction surgery, joint replacement surgery,hernia repair surgery, artificial vascular graft surgery, stentplacement, and neurosurgery are particularly prone to this complication.In each case, the implant becomes encapsulated by a fibrous connectivetissue capsule which compromises or impairs the function of the surgicalimplant (e.g., breast implant, artificial joint, surgical mesh, vasculargraft, stent or dural patch).

Adhesions generally begin to form within the first several days aftersurgery. Generally, adhesion formation is an inflammatory reaction inwhich factors are released, increasing vascular permeability andresulting in fibrinogen influx and fibrin deposition. This depositionforms a protein matrix that bridges the abutting tissues. Fibroblastsaccumulate, attach to the protein matrix, deposit collagen and induceangiogenesis. If this cascade of events can be prevented within 4 to 5days following surgery, then adhesion formation may be inhibited.

The compositions of the invention may be used to prevent adhesionformation in a wide variety of surgical procedures including spinal andneurosurgical procedures (e.g., open surgical resection of a rupturedlumbar disc or entrapped spinal nerve root (laminectomy); disectomies;and microlumbar disc excision (microdiscectomy)); gynecological surgicalprocedures (e.g., hysterectomy, myomectomy, endometriosis, infertility,birth control (e.g., tubal ligation), reversal of sterilization, pain,dysmennorrhea, dysfunctional uterine bleeding, ectopic pregnancy,ovarian cysts, and gynecologic malignancies); abdominal surgicalprocedures (e.g., hernia repair (abdominal, ventral, inguinal,incisional), bowel obstruction, inflammatory bowel disease (ulcerativecolitis, Crohn's disease), appendectomy, trauma (penetrating wounds,blunt trauma), tumor resection, infections (abscesses, peritonitis),cholecystectomy, gastroplasty (bariatric surgery), esophageal andpyloric strictures, colostomy, diversion iliostomy, anal-rectalfistulas, hemorrhoidectomies, splenectomy, hepatic tumor resection,pancreatitis, bowel perforation, upper and lower GI bleeding, andischemic bowel); cardiac surgical procedure (e.g., transplant surgery,vascular repair, coronary artery bypass grafting (CABG), congenitalheart defects, and valve replacements, staged procedures andreoperations (particularly repeat CABG surgery)); orthopedic surgicalprocedures (e.g., surgical interventions performed as a result of injuryor trauma (e.g., fractures (open and closed), sprains, jointdislocations, crush injuries, ligament and muscle tears, tendoninjuries, nerve injuries, congenital deformities and malformations,total joint or partial joint replacement, and cartilage injuries); andcosmetic or reconstructive surgical procedure (e.g., breastaugmentation, breast reconstruction after cancer surgery, craniofacialprocedures, reconstruction after trauma, congenital craniofacialreconstruction and oculoplastic surgical procedures).

For certain applications compositions may be include and/or release atherapeutic agent able to reduce scarring (i.e., a fibrosis-inhibitingagent) at a surgical site, such as to prevent or inhibit the formationof post-operative adhesions. Within one embodiment of the invention,compositions for the prevention of surgical adhesions may include or beadapted to release an agent that inhibits one or more of the fivegeneral components of the process of fibrosis (or scarring), including:inflammatory response and inflammation, migration and proliferation ofconnective tissue cells (such as fibroblasts or smooth muscle cells),formation of new blood vessels (angiogenesis), deposition ofextracellular matrix (ECM), and remodeling (maturation and organizationof the fibrous tissue). By inhibiting one or more of the components offibrosis (or scarring), the overgrowth of scar tissue at a surgical sitemay be inhibited or reduced.

Examples of fibrosis-inhibiting agents that may be combined with thepresent compositions to prevent the formation of adhesions include thefollowing: cell cycle inhibitors including (A) anthracyclines (e.g.,doxorubicin and mitoxantrone), (B) taxanes (e.g., paclitaxel, TAXOTERE®and docetaxel), and (C) podophyllotoxins (e.g., etoposide); (D)immunomodulators (e.g., sirolimus, everolimus, tacrolimus); (E) heatshock protein 90 antagonists (e.g., geldanamycin, 17-AAG, 17-DMAG); (F)HMGCoA reductase inhibitors (e.g., simvastatin); (G) inosinemonophosphate dehydrogenase inhibitors (e.g., mycophenolic acid,1-alpha-25 dihydroxy vitamin D₃); (H)NF kappa B inhibitors (e.g., Bay11-7082); (I) antimycotic agents (e.g., sulconizole) and (J) p38 MAPkinase inhibitors (e.g., SB202190), as well as analogues and derivativesof the aforementioned.

The drug dose administered from the present compositions for surgicaladhesion prevention will depend on a variety of factors, including thetype of formulation, the location of the treatment site, and the type ofcondition being treated; however, certain principles can be applied inthe application of this art. Drug dose can be calculated as a functionof dose per unit area (of the treatment site), total drug doseadministered can be measured, and appropriate surface concentrations ofactive drug can be determined Drugs are to be used at concentrationsthat range from several times more than to 50%, 20%, 10%, 5%, or evenless than 1% of the concentration typically used in a single systemicdose application. In certain aspects, the anti-scarring agent isreleased from the polymer composition in effective concentrations in atime period that may be measured from the time of infiltration intotissue adjacent to the device, which ranges from about less than 1 dayto about 180 days. Generally, the release time may also be from aboutless than 1 day to about 180 days; from about 7 days to about 14 days;from about 14 days to about 28 days; from about 28 days to about 56days; from about 56 days to about 90 days; from about 90 days to about180 days. In one aspect, the drug is released in effectiveconcentrations for a period ranging from 1-90 days.

The exemplary anti-fibrosing agents, used alone or in combination,should be administered under the following dosing guidelines. The totalamount (dose) of anti-scarring agent in the composition can be in therange of about 0.01 μg-10 μg, or 10 μg-10 mg, or 10 mg-250 mg, or 250mg-1000 mg, or 1000 mg-2500 mg. The dose (amount) of anti-scarring agentper unit area of surface to which the agent is applied may be in therange of about 0.01 μg/mm²-1 μg/mm², or 1 μg/mm²-10 μg/mm², or 10μg/mm²-250 μg/mm², 250 μg/mm²-1000 μg/mm², or 1000 μg/mm²-2500 μg/mm²

Provided below are exemplary dosage ranges for various anti-scarringagents that can be used in conjunction with compositions for treating orpreventing surgical adhesions in accordance with the invention. (A) Cellcycle inhibitors including doxorubicin and mitoxantrone. Doxorubicinanalogues and derivatives thereof: total dose not to exceed 25 mg (rangeof 0.1 μg to 25 mg); preferred 1 μg to 5 mg. Dose per unit area of 0.01μg-100 μg per mm²; preferred dose of 0.1 μg/mm²-10 μg/mm² Mitoxantroneand analogues and derivatives thereof: total dose not to exceed 5 mg(range of 0.01 μg to 5 mg); preferred 0.1 μg to 1 mg. Dose per unit areaof 0.01 μg-20 μg per mm²; preferred dose of 0.05 μg/mm²-3 μg/mm² (B)Cell cycle inhibitors including paclitaxel and analogues and derivatives(e.g., docetaxel) thereof: total dose not to exceed 10 mg (range of 0.1μg to 10 mg); preferred 1 μg to 3 mg. Dose per unit area of 0.1 μg-10 μgper mm²; preferred dose of 0.25 μg/mm²-5 μg/mm² (C) Cell cycleinhibitors such as podophyllotoxins (e.g., etoposide): total dose not toexceed 10 mg (range of 0.1 μg to 10 mg); preferred 1 μg to 3 mg. Doseper unit area of 0.1 μg-10 μg per mm²; preferred dose of 0.25 μg/mm²-5μg/mm² (D) Immunomodulators including sirolimus and everolimus.Sirolimus (i.e., rapamycin, RAPAMUNE): total dose not to exceed 10 mg(range of 0.1 μg to 10 mg); preferred 10 μg to 1 mg. Dose per unit areaof 0.1 μg-100 μg per mm²; preferred dose of 0.5 μg/mm²-10 μg/mm²Everolimus and derivatives and analogues thereof: total dose should notexceed 10 mg (range of 0.1 μg to 10 mg); preferred 10 μg to 1 mg. Doseper unit area of 0.1 μg-100 μg per mm² of surface area; preferred doseof 0.3 μg/mm²-10 μg/mm². (E) Heat shock protein 90 antagonists (e.g.,geldanamycin) and analogues and derivatives thereof: total dose not toexceed 20 mg (range of 0.1 μg to 20 mg); preferred 1 μg to 5 mg. Doseper unit area of 0.1 μg-10 μg per mm²; preferred dose of 0.25 μg/mm²-5μg/mm². (F) HMGCoA reductase inhibitors (e.g., simvastatin) andanalogues and derivatives thereof: total dose not to exceed 2000 mg(range of 10.0 μg to 2000 mg); preferred 10 μg to 300 mg. Dose per unitarea of 1.0 μg-1000 μg per mm²; preferred dose of 2.5 μg/mm²-500 μg/mm².(G) Inosine monophosphate dehydrogenase inhibitors (e.g., mycophenolicacid, 1-alpha-25 dihydroxy vitamin D₃) and analogues and derivativesthereof: total dose not to exceed 2000 mg (range of 10.0 μg to 2000 mg);preferred 10 μg to 300 mg. Dose per unit area of 1.0 μg-1000 μg per mm²;preferred dose of 2.5 μg/mm²-500 μg/mm². (H)NF kappa B inhibitors (e.g.,Bay 11-7082) and analogues and derivatives thereof: total dose not toexceed 200 mg (range of 1.0 μg to 200 mg); preferred 1 μg to 50 mg. Doseper unit area of 1.0 μg-100 μg per mm²; preferred dose of 2.5 μg/mm²-50μg/mm². (I) Antimycotic agents (e.g., sulconizole) and analogues andderivatives thereof: total dose not to exceed 2000 mg (range of 10.0 μgto 2000 mg); preferred 10 μg to 300 mg. Dose per unit area of 1.0μg-1000 μg per mm²; preferred dose of 2.5 μg/mm²-500 μg/mm² and (J) p38MAP kinase inhibitors (e.g., SB202190) and analogues and derivativesthereof: total dose not to exceed 2000 mg (range of 10.0 μg to 2000 mg);preferred 10 μg to 300 mg. Dose per unit area of 1.0 μg-1000 μg per mm²;preferred dose of 2.5 μg/mm²-500 μg/mm².

In a general method for coating tissues to prevent the formation ofadhesions following surgery, the multifunctional compounds are exposedto the modified environment and a thin layer of the composition is thenapplied to the tissues comprising, surrounding, and/or adjacent to thesurgical site before substantial reaction has occurred. Application ofthe multifunctional compounds to the tissue site may be by extrusion,brushing, spraying (as described above), or by any other convenientmeans. Thus, the invention also relates to a method of preventingadhesions between tissues of a patient, where steps (a) and (b) are asdescribed for the method of sealing tissue, and step (c) involvesallowing a three-dimensional matrix to form on the tissue, and thusprevent tissue adhesion.

Following application of the compounds to the surgical site,inter-reaction is allowed to continue in situ prior to closure of thesurgical incision. Once the reaction has reached equilibrium, tissuesthat are brought into contact with the coated tissues will not adherethereto. The surgical site can then be closed using conventional meanssuch as by sutures, and so forth.

In general, compounds that achieve complete inter-reaction within arelatively short period of time (i.e., 5-15 minutes following exposureof the multifunctional compounds to the modified environment) arepreferred for use in the prevention of surgical adhesions, so that thesurgical site may be closed relatively soon after completion of thesurgical procedure.

Certain surgical procedures may involve placement of a medical device orimplant at the surgical site, in which case it may be desirable to applythe composition (with or without a therapeutic agent) to the surface ofthe implant, to the implant-tissue interface, and/or to tissue in thevicinity of the implanted device to minimize the formation ofpost-operative surgical adhesions, unwanted scarring in the vicinity ofthe implant, and encapsulation of the implant by a fibrous connectivetissue capsule.

For the prevention of adhesions in spinal and neurosurgical procedures,the compositions alone or loaded with a therapeutic agent (e.g., afibrosis-inhibiting agent) may be applied to the tissue surface at aspinal or neurosurgical site or to the surface of an implanted device(e.g., dural patches, spinal prostheses, artificial disc, rods, bonefixation devices (e.g., anchoring plates and bone screws), injectablefilling or bulking agents for discs, spinal grafts, spinal nucleusimplants, intervertebral disc spacers, fusion cages, or to implantsplaced in the brain, such as drains, shunts, drug-delivery pumps, orneurostimulation devices) and/or the tissue surrounding the implantbefore, during, or after the surgical procedure.

For the prevention of adhesions associated with gynecologicalprocedures, the compositions alone or loaded with a therapeutic agent(e.g., a fibrosis-inhibiting agent) may be applied during open orendoscopic gynecological surgery to the tissue surface of the pelvicside wall, adnexa, uterus and any adjacent affected tissues during thesurgical procedure or to the surface of an implanted device or implant(e.g., genital-urinary stents, bulking agents, sterilization devices(e.g., valves, clips and clamps), and tubal occlusion implants andplugs) and/or the tissue surrounding the implant before, during, orafter the surgical procedure.

For the prevention of adhesions associated with abdominal surgicalprocedures, the compositions alone or loaded with a therapeutic agent(e.g., a fibrosis-inhibiting agent) may be applied during open,endoscopic, or laparoscopic abdominal surgery to the tissue surface ofthe peritoneal cavity, visceral peritoneum, abdominal organs, abdominalwall and any adjacent affected tissues during the surgical procedure orto the surface of an implanted device or implant and/or the tissuesurrounding the implant before, during, or after the surgical procedure.Representative examples of implants for use in abdominal proceduresincludes, without limitation, hernia meshes, restriction devices forobesity, implantable sensors, implantable pumps, peritoneal dialysiscatheters, peritoneal drug-delivery catheters, GI tubes for drainage orfeeding, portosystemic shunts, shunts for ascites, gastrostomy orpercutaneous feeding tubes, jejunostomy endoscopic tubes, colostomydevices, drainage tubes, biliary T-tubes, hemostatic implants, enteralfeeding devices, colonic and biliary stents, low profile devices,gastric banding implants, capsule endoscopes, anti-reflux devices, andesophageal stents.

For the prevention of adhesions associated with cardiac surgicalprocedures, the compositions alone or loaded with a therapeutic agent(e.g., a fibrosis-inhibiting agent) may be applied during open orendoscopic heart surgery to the tissue surface of the pericardium (orinfiltrated into the pericardial sac), heart, great vessels, pleura,lungs, chest wall and any adjacent affected tissues during the surgicalprocedure or to the surface of an implanted device or implant and/or thetissue surrounding the implant before, during, or after the surgicalprocedure. Representative examples of implants for use in cardiacprocedures includes, without limitation, heart valves (porcine,artificial), ventricular assist devices, cardiac pumps, artificialhearts, stents, bypass grafts (artificial and endogenous), patches,cardiac electrical leads, defibrillators and pacemakers.

For the prevention of adhesions associated with orthopedic surgicalprocedures, the compositions alone or loaded with a therapeutic agent(e.g., a fibrosis-inhibiting agent) may be applied during open orarthroscopic orthopedic surgery to the tissue surface of the bone,joint, muscle, tendon, ligament, cartilage and any adjacent affectedtissues during the surgical procedure or to the surface of an implantedorthopedic device or implant and/or the tissue surrounding the implantbefore, during, or after the surgical procedure. Representative examplesof implants for use in orthopedic procedures include plates, rods,screws, pins, wires, total and partial joint prostheses (artificialhips, knees, shoulders, phalangeal joints), reinforcement patches,tissue fillers, synthetic bone fillers, bone cement, synthetic graftmaterial, allograft material, autograft material, artificial discs,spinal cages, and intermedulary rods.

For the prevention of adhesions associated with cosmetic orreconstructive surgical procedures, the compositions alone or loadedwith a therapeutic agent (e.g., a fibrosis-inhibiting agent) may beapplied during open or endoscopic cosmetic surgery to the soft tissueimplant surface before, during, or after the implantation procedure orto the surface of the tissue of the implantation pocket immediatelyprior to, or during implantation of the soft tissue implant.Representative examples of soft tissue implants for use in cosmetic,plastic, and reconstructive surgical procedures include face, nose,breast, chin, buttocks, chest, lip and cheek implants) or to the surfaceof the soft tissue implant and/or the tissue surrounding the implantbefore, during, or after implantation of the soft tissue implant.

H. Implants and Coating Material for Implants

The multifunctional compounds of the invention can also be formed assolid implants, a term that is used herein to refer to any solid objectwhich is designed for insertion and use within the body, and includesbone and cartilage implants (e.g., artificial joints, retaining pins,cranial plates, and the like, of metal, plastic and/or other materials),breast implants (e.g., silicone gel envelopes, foam forms, and thelike), catheters and cannulas intended for long-term use (beyond aboutthree days) in place, artificial organs and vessels (e.g., artificialhearts, pancreases, kidneys, blood vessels, and the like), drug deliverydevices (including monolithic implants, pumps and controlled releasedevices such as ALZET® minipumps (Durect Corporation, Cupertino,Calif.), steroid pellets for anabolic growth or contraception, and thelike), sutures for dermal or internal use, periodontal membranes,ophthalmic shields, corneal lenticules, and the like.

Another use of the compounds is as a coating material for syntheticimplants. In a general method for coating a surface of a syntheticimplant, the multifunctional compounds are exposed to the modifiedenvironment, and a thin layer of the composition is then applied to asurface of the implant before substantial inter-reaction has occurred.In one embodiment, in order to minimize cellular and fibrous reaction tothe coated implant, the compounds are selected so as to result in amatrix that has a net neutral charge. Application of the compounds tothe implant surface may be by extrusion, brushing, spraying, or by anyother convenient means. Following application of the compounds to theimplant surface, inter-reaction is allowed to continue until completeand the three-dimensional matrix is formed.

Although this method can be used to coat the surface of any type ofsynthetic implant, it is particularly useful for implants where reducedthrombogenicity is an important consideration, such as artificial bloodvessels and heart valves, vascular grafts, vascular stents, andstent/graft combinations. The method may also be used to coatimplantable surgical membranes (e.g., monofilament polypropylene) ormeshes (e.g., for use in hernia repair). Breast implants may also becoated using the above method in order to minimize capsular contracture.

The compounds can also be coated on a suitable fibrous material, whichcan then be wrapped around a bone to provide structural integrity to thebone. The term “suitable fibrous material” as used herein, refers to afibrous material which is substantially insoluble in water,non-immunogenic, biocompatible, and immiscible with the crosslinkablecompositions of the invention. The fibrous material may comprise any ofa variety of materials having these characteristics and may be combinedwith crosslinkable compositions herein in order to form and/or providestructural integrity to various implants or devices used in connectionwith medical and pharmaceutical uses.

The compounds of the present invention may also be used to coatlenticules, which are made from either naturally occurring or syntheticpolymers.

I. Medical Implants Combined with Fibrosing Agents

In one aspect, medical implants may contain and/or are adapted torelease an agent which induces or promotes adhesion between the implantand tissue or a fibrotic reaction. The clinical performance of numerousmedical devices may be improved by anchoring the device effectively intothe surrounding tissue to provide either structural support or tofacilitate scarring and healing. Effective attachment of the device intothe surrounding tissue, however, is not always readily achieved. Onereason for ineffective attachment is that implantable medical devicesgenerally are composed of materials that are highly biocompatible anddesigned to reduce the host tissue response. These materials (e.g.,stainless steel, titanium based alloys, fluoropolymers, and ceramics)typically do not provide a good substrate for host tissue attachment andingrowth during the scarring process. As a result of poor attachmentbetween the device and the host tissue, devices can have a tendency tomigrate within the vessel or tissue in which they are implanted. Theextent to which a particular type of medical device can move or migrateafter implantation depends on a variety of factors including the typeand design of the device, the material(s) from which the device isformed, the mechanical attributes (e.g., flexibility and ability toconform to the surrounding geometry at the implantation site), thesurface properties, and the porosity of the device or device surface.The tendency of a device to loosen after implantation also depends onthe type of tissue and the geometry at the treatment site, where theability of the tissue to conform around the device generally can help tosecure the device in the implantation site. Device migration can resultin device failure and, depending on the type and location of the device,can lead to leakage, vessel occlusion, and/or damage to the surroundingtissue. Incorporation of a fibrosis-inducing agent with the compositionsof the invention can provide an effective, long-lasting andbiocompatible approach for anchoring implantable medical devices into oronto biological tissue.

In certain embodiments, the medical implant, when placed in to a tissue,releases an agent that induces or promotes adhesion between the implantand the tissue or a fibrotic reaction. In other embodiments, the medicalimplant contains or is made of a fibrosing agent, but does not releasethe fibrosing agent. In such embodiments, the fibrosing agent containedin the medical implant induces or promotes by direct contact of theagent to the tissue where the implant is placed.

Alternatively, or in addition, the tissue cavity into which the deviceor implant is placed can be treated with a fibrosis-inducing agent priorto, during, or after the implantation procedure. This can beaccomplished, for example, by topical application of the compositioncomprising a fibrosing agent or by spraying the composition into theanatomical space where the device can be placed or at the interfacebetween the implant and the tissue surface.

Representative examples of medical implants of particular utility foruse in combination with a fibrosis-inducing agent include, but are notrestricted to, orthopaedic implants (artificial joints, ligaments andtendons, screws, plates, and other implantable hardware), dentalimplants, intravascular implants (particularly arterial and venousocclusion devices and implants; vascular destructive implants), male andfemale contraceptive or sterilization devices and implants, soft palateimplants, embolization devices, surgical meshes (e.g., hernia repairmeshes, tissue scaffolds), fistula treatments, and spinal implants(e.g., artificial intervertebral discs, stent grafts, spinal fusiondevices, etc.).

As medical implants are made in a variety of configurations and sizes,the exact dose administered can vary with the amount injected or withthe device size, surface area and design; however, certain principlescan be applied in the application of this art. Drug dose can becalculated as a function of dose per unit area (of the portion of thedevice being coated), total drug dose administered can be measured, andappropriate surface concentrations of active drug can be determined. Itshould be readily evident to one of skill in the art that any of thepreviously described fibrosis inducing agents or derivatives oranalogues thereof can be utilized with the present compositions withoutdeviating from the spirit and scope of the invention.

Regardless of the method of application of the drug to the implant, theexemplary fibrosing agents, used alone or in combination, should beadministered under the following dosing guidelines:

Utilizing talc as an exemplary fibrosis-inducing agent, the total amountof talc delivered from an implant or coated onto the surface of animplant should not exceed 100 mg (range of 1 μg to 100 mg). In oneembodiment, the total amount of talc released from the implant should bein the range of 10 μg to 50 mg. The dose per unit area of the device(i.e., the dosage of talc as a function of the surface area of theportion of the device to which drug is applied and/or incorporated)should fall within the range of 0.05 μg-10 μg per mm² of surface areacoated. In another embodiment, talc should be applied to an implantsurface at a dose of 0.05 μg/mm²-10 μg/mm² of surface area coated.

Utilizing silk as an exemplary fibrosis-inducing agent, the total amountof silk delivered from an implant or coated onto the surface of animplant should not exceed 100 mg (range of 1 μg to 100 mg). In oneembodiment, the total amount of silk released from the prosthesis shouldbe in the range of 10 μg to 50 mg. The dose per unit area of the device(i.e., the dosage of silk as a function of the surface area of theportion of the device to which drug is applied and/or incorporated)should fall within the range of 0.05 μg-10 μg per mm² of surface areacoated. In another embodiment, silk should be applied to an implant at adose of 0.05 μg/mm²-10 μg/mm² of surface area coated.

Utilizing chitosan as an exemplary fibrosis-inducing agent, the totalamount of chitosan delivered from an implant or coated onto the surfaceof an implant, should not exceed 100 mg (range of 1 μg to 100 mg). Inone embodiment, the total amount of chitosan released from the implantshould be in the range of 10 μg to 50 mg. The dose per unit area of thedevice (i.e., the dosage of chitosan as a function of the surface areaof the portion of the device to which drug is applied and/orincorporated) should fall within the range of 0.05 μg-10 μg per mm² ofsurface area coated. In another embodiment, chitosan should be appliedto an implant surface at a dose of 0.05 μg/mm²-10 μg/mm² of surface areacoated.

Utilizing polylysine as an exemplary fibrosis-inducing agent, the totalamount polylysine delivered from an implant or coated onto the surfaceof an implant should not exceed 100 mg (range of 1 μg to 100 mg). In oneembodiment, the total amount of polylysine released from the implantshould be in the range of 10 μg to 50 mg. The dose per unit area of thedevice (i.e., the dosage of polylysine as a function of the surface areaof the portion of the device to which drug is applied and/orincorporated) should fall within the range of 0.05 μg-10 μg per mm² ofsurface area coated. In another embodiment, polylysine should be appliedto an implant surface at a dose of 0.05 μg/mm²-10 μg/mm² of surface areacoated.

Utilizing fibronectin as an exemplary fibrosis-inducing agent, the totalamount of fibronectin delivered from an implant or coated onto thesurface of an implant, should not exceed 100 mg (range of 1 μg to 100mg). In one embodiment, the total amount of fibronectin released fromthe prosthesis should be in the range of 10 μg to 50 mg. The dose perunit area of the device (i.e., the dosage of fibronectin as a functionof the surface area of the portion of the device to which drug isapplied and/or incorporated) should fall within the range of 0.05 μg-10μg per mm² of surface area coated. In another embodiment, talc should beapplied to an implant surface at a dose of 0.05 μg/mm²-10 μg/mm² ofsurface area coated.

Utilizing bleomycin as an exemplary fibrosis-inducing agent, the totalamount of bleomycin delivered from an implant, or coated onto thesurface of an implant, should not exceed 100 mg (range of 0.01 μg to 100mg). In one embodiment, the total amount of bleomycin released from theimplant should be in the range of 0.10 μg to 50 mg. The dose per unitarea of the device (i.e., the dosage of bleomycin as a function of thesurface area of the portion of the device to which drug is appliedand/or incorporated) should fall within the range of 0.005 μg-10 μg permm² of surface area coated. In another embodiment, bleomycin should beapplied to an implant surface at a dose of 0.005 μg/mm²-10 μg/mm² ofsurface area coated. In one embodiment, bleomycin is released from thesurface of an implant such that fibrosis in the tissue is promoted for aperiod ranging from several hours to several months.

Utilizing CTGF as an exemplary fibrosis-inducing agent, the total amountof CTGF delivered from an implant or coated onto the surface of animplant should not exceed 100 mg (range of 0.01 μg to 100 mg). In oneembodiment, the total amount of CTGF released from the implant should bein the range of 0.10 μg to 50 mg. The dose per unit area of the device(i.e., the dosage of CTGF as a function of the surface area of theportion of the device to which drug is applied and/or incorporated)should fall within the range of 0.005 μg-10 μg per mm² of surface areacoated. In another embodiment, CTGF should be applied to an implantsurface at a dose of 0.005 μg/mm²-10 μg/mm² of surface area coated.

The fibrosing agent (e.g., talc, silk, chitosan, polylysine,fibronectin, bleomycin, CTGF) may be released from the surface of theimplant such that fibrosis in the tissue is promoted for a periodranging from several hours to several months. For example, the fibrosingagent may be released in effective concentrations for a period rangingfrom 1 hour-30 days. It should be readily evident given the discussionsprovided herein that analogues and derivatives of the fibrosing agent(e.g., analogues and derivatives of talc, silk, chitosan, polylysine,fibronectin, bleomycin, CTGF, as previously described) with similarfunctional activity can be utilized for the purposes of this invention;the above dosing parameters are then adjusted according to the relativepotency of the analogue or derivative as compared to the parent compound(e.g., a compound twice as potent as the agent is administered at halfthe above parameters, a compound half as potent as the agent isadministered at twice the above parameters, etc.).

As described above, the device may additionally comprise an inflammatorycytokine (e.g., TGFβ, PDGF, VEGF, bFGF, TNFα, NGF, GM-CSF, IGF-a, IL-1,IL-1-13, IL-8, IL-6, and growth hormone) and/or a bone morphogenicprotein (BMP) (e.g., BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP-7 or ananalogue or derivative thereof).

Bone morphogenic protein(s) (e.g., BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, orBMP-7 or an analogue or derivative thereof) are to be used informulations at concentrations that range from 0.001 μg/mL toapproximately 20 mg/mL depending on the specific clinical application,formulation type (e.g., gel, liquid, solid, semi-solid), formulationchemistry, duration of required application, type of medical deviceinterface and formulation volume and or surface area coverage required.Preferably, the bone morphogenic protein is released in effectiveconcentrations for a period ranging from 1-180 days. The total dose fora single application is typically not to exceed 500 mg (range of 0.001μg to 500 mg); preferred 1 μg to 250 mg. When used as a device coating,the dose is per unit area of 0.001 μg-1000 μg per mm²; with a preferreddose of 0.01 μg/mm²-200 μg/mm². Minimum concentration of 10⁻⁹-10⁻⁴ M ofbone morphogenic protein is to be maintained on the device surface.

Inflammatory cytokines are to be used in formulations at concentrationsthat range from 0.0001 μg/mL to approximately 20 mg/mL depending on thespecific clinical application, formulation type (e.g., gel, liquid,solid, semi-solid), formulation chemistry, duration of requiredapplication, type of medical device interface and formulation volume andor surface area coverage required. Preferably, the inflammatory cytokineis released in effective concentrations for a period ranging from 1-180days. The total dose for a single application is typically not to exceed500 mg (range of 0.0001 μg to 100 mg); preferred 0.001 μg to 50 mg. Whenused as a device coating, the dose is per unit area of 0.0001 μg-500 μgper mm²; with a preferred dose of 0.001 μg/mm²-200 μg/mm². Minimumconcentration of 10⁻¹⁰-10⁻⁴ g/mL of inflammatory cytokine is to bemaintained on the device surface.

Furthermore, the device may alone or additionally comprise an agent thatstimulates cellular proliferation. Examples include: dexamethasone,isotretinoin (13-cis retinoic acid), 17-β-estradiol, estradiol, 1-α-25dihydroxyvitamin D₃, diethylstibesterol, cyclosporine A, L-NAME,all-trans retinoic acid (ATRA), and analogues and derivatives thereof.Doses used are those concentrations which are demonstrated to stimulatecell proliferation. The proliferative agents are to be used informulations at concentrations that range from 0.1 ng/mL to 25 mg/mLdepending on the specific clinical application, formulation type,formulation chemistry, duration of required application, type of medicaldevice interface and formulation volume, and/or surface area coveragerequired. Preferably, the proliferative agent is released in effectiveconcentrations for a period ranging from 1-180 days. The total dose fora single application is typically not to exceed 500 mg (range of 0.0001μg to 200 mg); preferred 0.001 μg to 100 mg. When used as a devicecoating, the dose is per unit area of 0.00001 μg-500 μg per mm²; with apreferred dose of 0.0001 μg/mm²-200 μg/mm². Minimum concentration of10⁻¹¹-10⁻⁶ M of proliferative agent is to be maintained on the devicesurface.

J. Medical Implants Combined with Fibrosis-Inhibiting Agents

In another aspect, medical implants may be coated with, or otherwiseadapted to release or incorporate an agent which inhibits the formationof reactive scar tissue on, or around, the surface of the device orimplant. Compositions that include a fibrosis-inhibiting agent may beused in combination with a variety of medical implants to make themresistant to overgrowth by inflammatory and fibrous scar tissue uponimplantation. Compositions and methods are described for coating medicaldevices and implants with drug-delivery compositions such that thepharmaceutical agent is delivered in therapeutic levels over a periodsufficient to allow normal healing to occur.

Upon implantation, excessive scar tissue growth can occur around the allor parts of the implant, which can lead to a reduction in theperformance of these devices. In certain cases, an implanted device maybe combined with a therapeutic agent (e.g., an anti-fibrotic agent) tominimize the formation of post-operative surgical adhesions, unwantedscarring in the vicinity of the implant, and encapsulation of theimplant by a fibrous connective tissue capsule.

Examples of medical devices of particular utility for use in combinationwith a fibrosis-inhibiting agent include, but are not restricted to,vascular stents, gastrointestinal stents, tracheal/bronchial stents,genital-urinary stents, ENT stents, intraocular lenses, implants forhypertrophic scars and keloids, vascular grafts, anastomotic connectordevices, surgical adhesion barriers, glaucoma drainage devices, film ormesh, prosthetic heart valves, tympanostomy tubes, penile implants,endotracheal and tracheostomy tubes, peritoneal dialysis catheters,intracranial pressure monitors, vena cava filters, central venouscatheters, ventricular assist devices (e.g., LVAD's), spinal prostheses,and gastrointestinal drainage tubes.

In one aspect, the medical device may be an electrical device (e.g., adevice having electrical components that can be placed in contact withtissue in an animal host and can provide electrical excitation tonervous or muscular tissue). Electrical devices can generate electricalimpulses and may be used to treat many bodily dysfunctions and disordersby blocking, masking, or stimulating electrical signals within the body.Electrical medical devices of particular utility in the presentinvention include, but are not restricted to, devices used in thetreatment of cardiac rhythm abnormalities, pain relief, epilepsy,Parkinson's Disease, movement disorders, obesity, depression, anxietyand hearing loss. Other examples of electrical devices includeneurostimulator and neurostimulation devices (e.g., electrical devicesfor electrical excitation of the central, autonomic, or peripheralnervous system), cardiac stimulation device such as cardiac rhythmmanagement devices, cardiac pacemakers, implantable cardiacdefibrillators (ICD) and other electrical devices for electricalexcitation of cardiac muscle tissue (including the specialized cardiacmuscle cells that make up the conductive pathways of the heart).Electrical devices also include electrical leads which are used as aconductor to carry electrical signals from the generator to the tissues.The electrical lead may be a wire or other material that transmitselectrical impulses from a generator (e.g., pacemaker, defibrillator, orother neurostimulator). Electrical leads may be unipolar, in which theyare adapted to provide effective therapy with only one electrode.Multi-polar leads are also available, including bipolar, tripolar andquadripolar leads.

In another aspect, the medical device may be an implantable sensor(i.e., a medical device that is implanted in the body to detect blood ortissue levels of a particular chemical (e.g., glucose, electrolytes,drugs, hormones) and/or changes in body chemistry, metabolites,function, pressure, flow, physical structure, electrical activity orother variable parameter). Representative examples of implantablesensors include, blood/tissue glucose monitors, electrolyte sensors,blood constituent sensors, temperature sensors, pH sensors, opticalsensors, amperometric sensors, pressure sensors, biosensors, sensingtransponders, strain sensors, activity sensors and magnetoresistivesensors.

In another aspect, the medical device may be a drug-delivery pump (i.e.,a medical device that includes a pump which is configured to deliver abiologically active agent (e.g., a drug) at a regulated dose). Thesedevices are implanted within the body and may include an externaltransmitter for programming the controlled release of drug, oralternatively, may include an implantable sensor that provides thetrigger for the drug delivery pump to release drug as physiologicallyrequired. Drug-delivery pumps may be used to deliver virtually anyagent, but specific examples include insulin for the treatment ofdiabetes, medication for the relief of pain, chemotherapy for thetreatment of cancer, anti-spastic agents for the treatment of movementand muscular disorders, or antibiotics for the treatment of infections.Representative examples of drug delivery pumps for use in the practiceof the invention include, without limitation, constant flow drugdelivery pumps, programmable drug delivery pumps, intrathecal pumps,implantable insulin delivery pumps, implantable osmotic pumps, oculardrug delivery pumps and implants, metering systems, peristaltic (roller)pumps, electronically driven pumps, elastomeric pumps,spring-contraction pumps, gas-driven pumps (e.g., induced byelectrolytic cell or chemical reaction), hydraulic pumps,piston-dependent pumps and non-piston-dependent pumps, dispensingchambers, infusion pumps, passive pumps, infusate pumps andosmotically-driven fluid dispensers.

In yet another aspect, the medical device may be a soft tissue implant.Soft tissue implants are medical devices that may includes a volumereplacement material for augmentation or reconstruction to replace awhole or part of a living structure. Soft tissue implants are used forthe reconstruction of surgically or traumatically created tissue voids,augmentation of tissues or organs, contouring of tissues, therestoration of bulk to aging tissues, and to correct soft tissue foldsor wrinkles (rhytides). Soft tissue implants may be used for theaugmentation of tissue for cosmetic (aesthetic) enhancement or inassociation with reconstructive surgery following disease or surgicalresection. Representative examples of soft tissue implants includebreast implants, chin implants, calf implants, cheek implants and otherfacial implants, buttocks implants, and nasal implants.

Soft tissue implants that release a therapeutic agent for reducingscarring at the implant-tissue interface can be used to enhance theappearance, increase the longevity, reduce the need for correctivesurgery or repeat procedures, decrease the incidence of pain and othersymptoms, and improve the clinical function of implant. Accordingly, thepresent invention provides soft tissue implants that are coated orotherwise incorporate an anti-scarring agent or a composition thatincludes an anti-scarring agent.

According to the present invention, any fibrosis-inhibiting agentdescribed above can be utilized in the practice of this embodiment.Within one embodiment of the invention, medical implants may be adaptedto release an agent that inhibits one or more of the four generalcomponents of the process of fibrosis (or scarring), including:formation of new blood vessels (angiogenesis), migration andproliferation of connective tissue cells (such as fibroblasts or smoothmuscle cells), deposition of extracellular matrix (ECM), and remodeling(maturation and organization of the fibrous tissue). By inhibiting oneor more of the components of fibrosis (or scarring), the overgrowth ofgranulation tissue may be inhibited or reduced.

Several examples of agents for use with medical implants include thefollowing: cell cycle inhibitors including (A) anthracyclines (e.g.,doxorubicin and mitoxantrone), (B) taxanes (e.g., paclitaxel, TAXOTEREand docetaxel), and (C) podophyllotoxins (e.g., etoposide); (D)immunomodulators (e.g., sirolimus, everolimus, tacrolimus); (E) heatshock protein 90 antagonists (e.g., geldanamycin); (F) HMGCoA reductaseinhibitors (e.g., simvastatin); (G) inosine monophosphate dehydrogenaseinhibitors (e.g., mycophenolic acid, 1-alpha-25 dihydroxy vitamin D₃);(H)NF kappa B inhibitors (e.g., Bay 11-7082); (I) antimycotic agents(e.g., sulconizole), (J) p38 MAP kinase inhibitors (e.g., SB202190), and(K) angiogenesis inhibitors (e.g., halofuginone bromide), as well asanalogues and derivatives of the aforementioned.

Regardless of the method of application of the drug to the device, theexemplary anti-fibrosing agents, used alone or in combination, should beadministered under the following dosing guidelines. The total amount(dose) of anti-scarring agent in or on the device may be in the range ofabout 0.01 μg-10 μg, or 10 μg-10 mg, or 10 mg-250 mg, or 250 mg-1000 mg,or 1000 mg-2500 mg. The dose (amount) of anti-scarring agent per unitarea of device surface to which the agent is applied may be in the rangeof about 0.01 μg/mm²-1 μg/mm², or 1 μg/mm²-10 μg/mm², or 10 μg/mm²-250μg/mm², 250 μg/mm²-1000 μg/mm², or 1000 μg/mm²-2500 μg/mm².

As medical implants are made in a variety of configurations and sizes,the exact dose administered will vary with device size, surface area anddesign; however, certain principles can be applied in the application ofthis art. Drug dose can be calculated as a function of dose per unitarea (of the portion of the device being coated), total drug doseadministered, and appropriate surface concentrations of active drug canbe determined Drugs are to be used at concentrations that range fromseveral times more than to 10%, 5%, or even less than 1% of theconcentration typically used in a single chemotherapeutic systemic doseapplication. Preferably, the drug is released in effectiveconcentrations for a period ranging from 1-90 days.

Provided below are exemplary dosage ranges for various anti-scarringagents that can be used in conjunction with medical implants inaccordance with the invention. A) Cell cycle inhibitors includingdoxorubicin and mitoxantrone. Doxorubicin analogues and derivativesthereof: total dose not to exceed 25 mg (range of 0.1 μg to 25 mg);preferred 1 μg to 5 mg. The dose per unit area of 0.01 μg-100 μg permm²; preferred dose of 0.1 μg/mm²-10 μg/mm². Mitoxantrone and analoguesand derivatives thereof: total dose not to exceed 5 mg (range of 0.01 μgto 5 mg); preferred 0.1 μg to 1 mg. The dose per unit area of the deviceof 0.01 μg-20 μg per mm²; preferred dose of 0.05 μg/mm²-3 μg/mm². B)Cell cycle inhibitors including paclitaxel and analogues and derivatives(e.g., docetaxel) thereof: total dose not to exceed 10 mg (range of 0.1μg to 10 mg); preferred 1 μg to 3 mg. The dose per unit area of thedevice of 0.1 μg-10 μg per mm²; preferred dose of 0.25 μg/mm²-5 μg/mm².(C) Cell cycle inhibitors such as podophyllotoxins (e.g., etoposide):total dose not to exceed 10 mg (range of 0.1 μg to 10 mg); preferred 1μg to 3 mg. The dose per unit area of the device of 0.1 μg-10 μg permm²; preferred dose of 0.25 μg/mm²-5 μg/mm². (D) Immunomodulatorsincluding sirolimus and everolimus. Sirolimus (i.e., rapamycin,RAPAMUNE): Total dose not to exceed 10 mg (range of 0.1 μg to 10 mg);preferred 10 μg to 1 mg. The dose per unit area of 0.1 μg-100 μg permm²; preferred dose of 0.5 μg/mm²-10 μg/mm² Everolimus and derivativesand analogues thereof: Total dose should not exceed 10 mg (range of 0.1μg to 10 mg); preferred 10 μg to 1 mg. The dose per unit area of 0.1μg-100 μg per mm² of surface area; preferred dose of 0.3 μg/mm²-10μg/mm². (E) Heat shock protein 90 antagonists (e.g., geldanamycin) andanalogues and derivatives thereof: total dose not to exceed 20 mg (rangeof 0.1 μg to 20 mg); preferred 1 μg to 5 mg. The dose per unit area ofthe device of 0.1 μg-10 μg per mm²; preferred dose of 0.25 μg/mm²-5μg/mm². (F) HMGCoA reductase inhibitors (e.g., simvastatin) andanalogues and derivatives thereof: total dose not to exceed 2000 mg(range of 10.0 μg to 2000 mg); preferred 10 μg to 300 mg. The dose perunit area of the device of 1.0 μg-1000 μg per mm²; preferred dose of 2.5μg/mm²-500 μg/mm². (G) Inosine monophosphate dehydrogenase inhibitors(e.g., mycophenolic acid, 1-alpha-25 dihydroxy vitamin D₃) and analoguesand derivatives thereof: total dose not to exceed 2000 mg (range of 10.0μg to 2000 mg); preferred 10 μg to 300 mg. The dose per unit area of thedevice of 1.0 μg-1000 μg per mm²; preferred dose of 2.5 μg/mm²-500μg/mm². (H)NF kappa B inhibitors (e.g., Bay 11-7082) and analogues andderivatives thereof: total dose not to exceed 200 mg (range of 1.0 μg to200 mg); preferred 1 μg to 50 mg. The dose per unit area of the deviceof 1.0 μg-100 μg per mm²; preferred dose of 2.5 μg/mm²-50 μg/mm². (I)Antimycotic agents (e.g., sulconizole) and analogues and derivativesthereof: total dose not to exceed 2000 mg (range of 10.0 μg to 2000 mg);preferred 10 μg to 300 mg. The dose per unit area of the device of 1.0μg-1000 μg per mm²; preferred dose of 2.5 μg/mm²-500 μg/mm². (J) p38 MAPKinase Inhibitors (e.g., SB202190) and analogues and derivativesthereof: total dose not to exceed 2000 mg (range of 10.0 μg to 2000 mg);preferred 10 μg to 300 mg. The dose per unit area of the device of 1.0μg-1000 μg per mm²; preferred dose of 2.5 μg/mm²-500 μg/mm² (K)anti-angiogenic agents (e.g., halofuginone bromide) and analogues andderivatives thereof: total dose not to exceed 10 mg (range of 0.1 μg to10 mg); preferred 1 μg to 3 mg. The dose per unit area of the device of0.1 μg-10 μg per mm²; preferred dose of 0.25 μg/mm²-5 μg/mm²

In addition to those described above (e.g., sirolimus, everolimus, andtacrolimus), several other examples of immunomodulators and appropriatedosages ranges for use with medical implants include the following: (A)Biolimus and derivatives and analogues thereof: Total dose should notexceed 10 mg (range of 0.1 μg to 10 mg); preferred 10 μg to 1 mg. Thedose per unit area of 0.1 μg-100 μg per mm² of surface area; preferreddose of 0.3 μg/mm²-10 μg/mm². (B) Tresperimus and derivatives andanalogues thereof: Total dose should not exceed 10 mg (range of 0.1 μgto 10 mg); preferred 10 μg to 1 mg. The dose per unit area of 0.1 μg-100μg per mm² of surface area; preferred dose of 0.3 μg/mm²-μg/mm². (C)Auranofin and derivatives and analogues thereof: Total dose should notexceed 10 mg (range of 0.1 μg to 10 mg); preferred 10 μg to 1 mg. Thedose per unit area of 0.1 μg-100 μg per mm² of surface area; preferreddose of 0.3 μg/mm²-10 μg/mm². (D) 27-O-Demethylrapamycin and derivativesand analogues thereof: Total dose should not exceed 10 mg (range of 0.1μg to 10 mg); preferred 10 μg to 1 mg. The dose per unit area of 0.1μg-100 μg per mm² of surface area; preferred dose of 0.3 μg/mm²-10μg/mm². (E) Gusperimus and derivatives and analogues thereof: Total doseshould not exceed 10 mg (range of 0.1 μg to 10 mg); preferred 10 μg to 1mg. The dose per unit area of 0.1 μg-100 μg per mm² of surface area;preferred dose of 0.3 μg/mm²-10 μg/mm². (F) Pimecrolimus and derivativesand analogues thereof: Total dose should not exceed 10 mg (range of 0.1μg to 10 mg); preferred 10 μg to 1 mg. The dose per unit area of 0.1μg-100 μg per mm² of surface area; preferred dose of 0.3 μg/mm²-10μg/mm² and (G) ABT-578 and analogues and derivatives thereof: Total doseshould not exceed 10 mg (range of 0.1 μg to 10 mg); preferred 10 μg to 1mg. The dose per unit area of 0.1 μg-100 μg per mm² of surface area;preferred dose of 0.3 μg/mm²-10 μg/mm²

In a general method for coating a surface of a synthetic implant, thecomposition is exposed to the aqueous environment, and a thin layer ofthe composition is then applied to a surface of the implant beforesubstantial inter-reaction has occurred. In one embodiment, in order tominimize cellular and fibrous reaction to the coated implant, thecomponents are selected so as to result in a matrix that has a netneutral charge. Application of the composition to the implant surfacemay be by extrusion, brushing, spraying (as described above), or by anyother convenient means. Following application of the composition to theimplant surface, inter-reaction is allowed to continue until completeand the three-dimensional matrix is formed.

Although this method can be used to coat the surface of any type ofsynthetic implant, it is particularly useful for implants where reducedthrombogenicity is an important consideration, such as artificial bloodvessels and heart valves, vascular grafts, vascular stents, andstent/graft combinations. The method may also be used to coatimplantable surgical membranes (e.g., monofilament polypropylene) ormeshes (e.g., for use in hernia repair). Breast implants may also becoated using the above method in order to minimize capsular contracture.

The compositions of the invention can also be coated on a suitablefibrous material, which can then be wrapped around a bone to providestructural integrity to the bone. The term “suitable fibrous material”as used herein, refers to a fibrous material which is substantiallyinsoluble in water, non-immunogenic, biocompatible, and immiscible withthe crosslinkable compositions of the invention. The fibrous materialmay comprise any of a variety of materials having these characteristicsand may be combined with crosslinkable compositions herein in order toform and/or provide structural integrity to various implants or devicesused in connection with medical and pharmaceutical uses.

The compositions of the present invention may also be used to coatlenticules, which are made from either naturally occurring or syntheticpolymers.

I. Treatment of Aneurysm

The multifunctional compounds can be extruded or molded in the shape ofa string or coil, then dehydrated. The resulting dehydrated string orcoil can be delivered via catheter to the site of a vascularmalformation, such as an aneurysm, for the purpose of vascular occlusionand, ultimately, repair of the malformation. The dehydrated string orcoil can be delivered in a compact size and will rehydrate inside theblood vessel, swelling several times in size compared to its dehydratedstate, while maintaining its original shape.

Thus, another embodiment of the invention is a method for treating ananeurysm, where steps (a) and (b) are as described for the method ofsealing tissue, and step (c) involves allowing a three-dimensionalmatrix to form in the desired shape, delivering it to the site ofinterest, and allowing the matrix to rehydrate in situ.

J. Other Uses

As discussed in U.S. Pat. No. 5,752,974 to Rhee et al., themultifunctional compounds can be used to block or fill various lumensand voids in the body of a mammalian subject. The compounds can also beused as biosealants to seal fissures or crevices within a tissue orstructure (such as a vessel), or junctures between adjacent tissues orstructures, to prevent leakage of blood or other biological fluids. Thecompositions may also be used to seal or close a fistula, where ascar-promoting agent or sclerosing agent, e.g., silk, may be included inthe composition to promote tissue closure.

The multifunctional compounds can also be used as a large space-fillingdevice for organ displacement in a body cavity during surgical orradiation procedures, for example, to protect the intestines during aplanned course of radiation to the pelvis.

The compounds can also be coated onto the interior surface of aphysiological lumen, such as a blood vessel or Fallopian tube, therebyserving as a sealant to prevent restenosis of the lumen followingmedical treatment, such as, for example, balloon catheterization toremove arterial plaque deposits from the interior surface of a bloodvessel, or removal of scar tissue or endometrial tissue from theinterior of a Fallopian tube. A thin layer of the compounds ispreferably applied to the interior surface of the vessel (for example,via catheter) immediately following exposure to the modifiedenvironment. Because the compounds of the invention are not readilydegradable in vivo, the potential for restenosis due to degradation ofthe coating is minimized. The use of a three-dimensional matrix having anet neutral charge further minimizes the potential for restenosis.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description, as well as the examples that are presented above,are intended to illustrate and not limit the scope of the invention.Other aspects, advantages, and modifications will be apparent to thoseskilled in the art to which the invention pertains. All patents, patentapplications, journal articles, and other references cited herein areincorporated by reference in their entireties.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compounds of the invention, and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C., and pressure is at or near atmospheric.

Example 1 Preparation of a Multifinctional Compound

0.1 g (˜2 eq) of NaOH is added to a solution of methanol (25 mL) and 10g of tetra-functional HS-PEG (Mw 10,000). The solution is stirred untilall of the NaOH is dissolved. The solution is then evaporated and theresidue is dissolved into methylene chloride (20 mL). The methylenechloride solution is then dried with MgSO₄. After the polymer solutionhas been filtered it is slowly dropped into a solution of methylenechloride with a 10 times excess of acrolyl chloride. The material isstirred at room temperature for a few hours and then filtered to removethe NaCl before it is precipitated into diethyl ether. The di-functionalHS— di-functional acrolyl PEG final product is then isolated as a whitepowder after filtration. This powder can then be dissolved in a 0.1 MHCl aqueous solution at a 40% concentration, which then can beco-extruded with a 0.3 M pH 9.6 buffer prepared from a mixture of sodiumcarbonate and sodium phosphate. The material will gel immediately uponcoextrusion.

Example 2 Gellation of a Multifinctional Compound

The multifunctional compound from Example 1 is dissolved in a 0.1 M HClaqueous solution at a 40% (w/v) concentration, which is then co-extrudedwith a 0.3 M pH 9.6 buffer, prepared from a mixture of sodium carbonateand sodium phosphate. The material gels immediately upon coextrusion.

Example 3 Preparation of Drug Loaded Microspheres by Spray Drying

3.6 grams of methoxy poly(ethylene glycol 5000))-block-(poly(DL-lactide) (65:35 MePEG:PDLLA weight ratio) was dissolved in 200 mLmethylene chloride. 400 mg of a drug (mycophenolic acid (MPA),chlorpromazine (CPZ) or paclitaxel (PTX)) was added and the resultingsolution was spray dried (Buchi spray drier model B 191). Inlettemperature 50° C., outlet temperature <39° C., aspirator 100%, flowrate 700 l/hr. The collected microspheres were dried under vacuum atroom temperature overnight to produce uniform, spherical particleshaving size ranges of less than about 10 microns (typically about 0.5 toabout 2 microns).

Example 4 MPA Loaded Microspheres (<10 Micron)

100 mL of freshly prepared 10% polyvinyl alcohol (PVA) solution and 10mL of pH 3 acetic acid solution saturated with MPA was added into a 600mL beaker. The acidified PVA solution was stirred at 2000 rpm for 30minutes. Meanwhile, a solution of 400 mg MPA and 800 mg MePEG5000-PDLLA(65:35) in 20 mL dichloromethane was prepared. Thepolymer/dichloromethane solution was added dropwise to the PVA solutionwhile stirring at 2000 rpm with a Fisher DYNA-MIX stirrer. Afteraddition was complete, the solution was allowed to stir for anadditional 45 minutes. The microsphere solution was transferred toseveral disposable graduated polypropylene conical centrifuge tubes,washed with pH 3 acetic acid solution saturated with MPA, andcentrifuged at 2600 rpm for 10 minutes. The aqueous layer was decantedand the washing, centrifuging and decanting was repeated 3 times. Thecombined, washed microspheres were freeze-dried and vacuum dried toremove any excess water.

Example 5 MPA Containing Microspheres (50-100 Micron)

Microspheres having an average size of about 50-100 microns wereprepared using a 1% PVA solution and 500 rpm stirring rate using thesame procedure described in Example 4.

Example 6 CPZ and PTX Containing Microspheres

Paclitaxel (PTX) and chlorpromazine (CPZ) containing microspheres wereprepared using the procedure described in Example 4 with the exceptionthat the PVA solution and the washing solution does not have to beacidified and saturated with the drug.

Example 7 Paclitaxel Containing Micelles

MePEG2000 (41 g) and MePEG2000-PDLLA (60:40) (410 g) were combined in avessel and heated to 75° C. with stirring. After the polymers werecompletely melted and mixed, the temperature was decreased to 55° C.Meanwhile, a PTX solution in tetrahydrofuran (46 g/200 mL) was preparedand poured into the polymer solution under constant stirring. Stirringwas continued for and additional hour. The PTX containing micelles weredried at 50° C. under vacuum to remove solvent and were ground on a 2 mmmesh screen after cooling.

Example 8 Surgical Adhesions Model to Assess Fibrosis Inhibiting Agentsin Rats

The rat caecal sidewall model is used to as to assess the anti-fibroticcapacity of formulations in vivo. Sprague Dawley rats are anesthetizedwith halothane. Using aseptic precautions, the abdomen is opened via amidline incision. The caecum is exposed and lifted out of the abdominalcavity. Dorsal and ventral aspects of the caecum are successivelyscraped a total of 45 times over the terminal 1.5 cm using a No. 10scalpel blade. Blade angle and pressure are controlled to producepunctate bleeding while avoiding severe tissue damage. The left side ofthe abdomen is retracted and everted to expose a section of theperitoneal wall that lies proximal to the caecum. The superficial layerof muscle (transverses abdominis) is excised over an area of 1×2 cm²,leaving behind torn fibers from the second layer of muscle (internaloblique muscle). Abraded surfaces are tamponaded until bleeding stops.The abraded caecum is then positioned over the sidewall wound andattached by two sutures. The formulation is applied over both sides ofthe abraded caecum and over the abraded peritoneal sidewall. A furthertwo sutures are placed to attach the caecum to the injured sidewall by atotal of 4 sutures and the abdominal incision is closed in two layers.After 7 days, animals are evaluated post mortem with the extent andseverity of adhesions being scored both quantitatively andqualitatively.

Example 9 Surgical Adhesions Model to Assess Fibrosis Inhibiting Agentsin Rabbits

The rabbit uterine horn model is used to assess the anti-fibroticcapacity of formulations in vivo. Mature New Zealand White (NZW) femalerabbits are placed under general anesthetic. Using aseptic precautions,the abdomen is opened in two layers at the midline to expose the uterus.Both uterine horns are lifted out of the abdominal cavity and assessedfor size on the French Scale of catheters. Horns between Nos. 8 and 14on the French Scale (2.5-4.5 mm diameter) are deemed suitable for thismodel. Both uterine horns and the opposing peritoneal wall are abradedwith a #10 scalpel blade at a 45° angle over an area 2.5 cm in lengthand 0.4 cm in width until punctuate bleeding is observed. Abradedsurfaces are tamponaded until bleeding stops. The individual horns arethen opposed to the peritoneal wall and secured by two sutures placed 2mm beyond the edges of the abraded area. The formulation is applied andthe abdomen is closed in three layers. After 14 days, animals areevaluated post mortem with the extent and severity of adhesions beingscored both quantitatively and qualitatively.

Example 10 Spinal Surgical Adhesions Model to Assess Fibrosis InhibitingAgents in Rabbits

Extensive scar formation and adhesions often occur after lumbar spinesurgery involving the vertebrae. The dense and thick fibrous tissueadherent to the spine and adjacent muscles must be removed by surgery.Unfortunately, fibrous adhesions usually reform after the secondarysurgery. Adhesions are formed by proliferation and migration offibroblasts from the surrounding tissue at the site of surgery. Thesecells are responsible for the healing response after tissue injury. Oncethey have migrated to the wound they lay down proteins such as collagento repair the injured tissue. Overproliferation and secretion by thesecells induce local obstruction, compression, and contraction of thesurrounding tissues with accompanying side effects.

The rabbit laminectomy spinal adhesion model described herein is used toinvestigate spinal adhesion prevention by local slow release ofantifibrotic drugs.

Five to six animals are included in each experimental group to allow formeaningful statistical analysis. Formulations with variousconcentrations of antifibrotic drugs are tested against control animalsto assess inhibition of adhesion formation.

Rabbits are anesthetized with an IM injection of ketamine/zylazine. Anendotracheal tube is inserted for maintenance of anesthesia withhalothane. The animal is placed prone on the operating table on top of aheating pad and the skin over the lower half of the back is shaved andprepared for sterile surgery. A longitudinal midline skin incision ismade from L-1 to L-5 and down the lumbosacral fascia. The fascia isincised to expose the tips of the spinous processes. The paraspinousmuscles are dissected and retracted from the spinous process and laminaof L-4. A laminectomy is performed at L-4 by removal of the spinalprocess with careful bilateral excision of the laminae, thus creating asmall 5×10 mm laminectomy defect. Hemostasis is obtained with Gelfoam.The test formulations are applied to the injury site and the wound isclosed in layers with Vicryl sutures. The animals are placed in anincubator until recovery from anesthesia and then returned to theircage.

Two weeks after surgery, the animals are anesthetized using proceduressimilar to those described above. The animals are euthanized withEuthanyl. After a skin incision, the laminectomy site is analyzed bydissection and the amount of adhesion is scored using scoring systemspublished in the scientific literature for this type of injury.

Example 11 Tendon Surgical Adhesions Model to Assess Fibrosis InhibitingAgents in Rabbits

This model is used to investigate whether adhesion of the tendons can beprevented by local slow release of drugs known to inhibit fibrosis.Polymeric formulations are loaded with drugs and implanted aroundinjured tendons in rabbits. In animals without fibrosis-inhibitingformulations, adhesions develop within 3 weeks of flexor tendon injuryif immobilization of the tendon is maintained during that period. Anadvantage of rabbits is that their tendon anatomy and cellular behaviourduring tendon healing are similar to those in man except for the rate ofhealing that is much faster in rabbits.

Rabbits are anesthetized and the skin over the right hindlimb is shavedand prepared for sterile surgery. Sterile surgery is performed aided byan operating microscope. A longitudinal midline skin incision is made onthe volvar aspect of the proximal phalange in digits 2 and 4. Thesynovial sheath of the tendons is carefully exposed and incisedtransversally to access the flexor digitorum profundus distal to theflexor digitorum superficialis bifurcation. Tendon injury is performedby gently lifting the flexor digitorum profundus with curved forceps andincising transversally through half of its substance. The formulationcontaining the test drug formulation is applied around the tendons inthe sheath of one of the two digits randomly selected. The other digitis left untreated and is used as a control. The sheath is then repairedwith 6-0 nylon suture. An immobilizing 6-0 nylon suture is insertedthrough the transverse metacarpal ligament into the tendon/sheathcomplex to immobilize the tendon and the sheath as a single unit toencourage adhesion formation. The wound is closed with 4-0 interruptedsutures. A bandage is applied around the hindpaw to further augmentimmobilization of the digits and ensure comfort and ambulation of theanimals. The animals are recovered and returned to their cage.

Three weeks after surgery, the animals are anesthetized. After a skinincision, the tissue plane around the synovial sheath is dissected andthe tendon-sheath complex harvested en block and transferred in 10%phosphate buffered formaldehyde for histopathology analysis. The animalsare then euthanized. After paraffin embedding, serial 5-um thincross-sections are cut every 2 mm through the sheath and tendon complex.Sections are stained with H&E and Movat's stains to evaluate adhesiongrowth. Each slide is digitized using a computer connected to a digitalmicroscope camera (Nikon Micropublisher cooled camera). Morphometryanalysis is then performed using image analysis software (ImagePro).Thickness and area of adhesion defined as the substance obliterating thesynovial space are measured and compared between formulation-treated andcontrol animals.

Example 12 Mycophenolic Acid in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg), and MPA (60mg, sifted <100 micron). A 1 mL capped syringe (syringe 2) is filledwith 0.25 mL of 6.3 mM HCl solution (pH 2.1). A 1 mL capped syringe(syringe 3) is filled with 0.35 mL 0.24 M monobasic sodium phosphate and0.4 M sodium carbonate (pH 10.0) buffer. The solid contents of syringe 1and the acidic solution of syringe 2 are mixed through a mixingconnector by repeatedly transferring the contents from one syringe tothe other. After complete mixing, the entire mixture is pushed into oneof the syringes. The syringe containing the mixture then is attached toone inlet of an applicator (MICROMEDICS® air assisted spray-applicator(Model SA-6105)). Syringe 3 containing the pH 9.7 solution is attachedonto the other inlet of the applicator. The formulation is applied to atissue surface as specified by the applicator manufacturer.

Example 13 Mycophenolic Acid and Disodium Salt of MPA (Na₂MPA) in aMultifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg). A 1 mLcapped syringe (syringe 2) is filled with 0.25 mL of 6.3 mM HCl solution(pH 2.1). A 1 mL capped syringe (syringe 3) is filled with 0.25 mL 0.12M monobasic sodium phosphate and 0.2 M sodium carbonate (pH 9.7) buffer.A 1 mL syringe (syringe 4) equipped with luer-lock mixing connector isfilled with MPA (5 mg) and Na₂MPA (95 mg), both sifted <100 micron. Thecontents of syringe 4 and syringe 2 are mixed through a mixing connectorby repeatedly transferring the contents from one syringe to the other.This solution is then used to reconstitute the solids in syringe 1.After complete mixing, all of the formulation is pushed into one of thesyringes, which is then attached to one inlet of an applicator(MICROMEDICS® air assisted spray-applicator (Model SA-6105)). Syringe 3containing the pH 9.7 solution is attached onto the other inlet of theapplicator. The formulation is applied to a tissue surface as specifiedby the applicator manufacturer.

Example 14 Chlorpromazine in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg), and CPZ (5or 10 mg). A 1 mL capped syringe (syringe 2) is filled with 0.25 mL of6.3 mM HCl solution (pH 2.1). A 1 mL capped syringe (syringe 3) isfilled 0.25 mL 0.12 M monobasic sodium phosphate and 0.2 M sodiumcarbonate (pH 9.7) buffer. The components are mixed and applied to atissue surface using the procedure described in Example 12.

Example 15 Paclitaxel-Loaded Microspheres in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg), and 10% PTXloaded MePEG5000-PDLLA (65:35) microspheres prepared by spray drying(0.5 or 2 mg) (prepared using the procedure described in Example 12). A1 mL capped syringe (syringe 2) is filled with 0.25 mL of 6.3 mM HClsolution (pH 2.1). A 1 mL capped syringe (syringe 3) is filled 0.25 mL0.12 M monobasic sodium phosphate and 0.2 M sodium carbonate (pH 9.7)buffer. The components are mixed and applied to a tissue surface usingthe procedure described in Example 12.

Example 16 CPZ-Loaded Microspheres in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg), and 10% CPZloaded MePEG5000-PDLLA (65:35) microspheres prepared by spray drying (50or 100 mg) (prepared using the procedure described in Example 12). A 1mL capped syringe (syringe 2) is filled with 0.25 mL of 6.3 mM HClsolution (pH 2.1). A 1 mL capped syringe (syringe 3) is filled 0.25 mL0.12 M monobasic sodium phosphate and 0.2 M sodium carbonate (pH 9.7)buffer. The components are mixed and applied to a tissue surface usingthe procedure described in Example 12.

Example 17 MPA-Loaded Microspheres in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg), and 10% MPAloaded MePEG5000-PDLLA 65:35 microspheres prepared by spray drying (25or 75 mg) (prepared using the procedure described in Example 13). A 1 mLcapped syringe (syringe 2) is filled with 0.25 mL 6.3 mM HCl solution(pH 2.1). A 1 mL capped syringe (syringe 3) iss filled 0.35 mL 0.24 Mmonobasic sodium phosphate and 0.4 M sodium carbonate (pH 10.0) buffer.The components are mixed and applied to a tissue surface using theprocedure described in Example 12.

Example 18 Incorporation of PTX Loaded Micelles into a MultifunctionalCompound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg). A 2 mLserum vial iss filled with 1.5 mL of 6.3 mM HCl solution (pH 2.1). A 1mL capped syringe (syringe 2) is filled with 0.25 mL 0.12 M monobasicsodium phosphate and 0.2 M sodium carbonate (pH 9.7) buffer. A 2 mLserum vial is filled with 10% PTX loaded micelles (2 mg or 8 mg)(prepared as in Example 7) and reconstituted with 1 mL of the pH 2.1solution. 0.25 mL of the micelle solution is removed with a 1 mLsyringe; the syringe iss attached to syringe 1 containing the solidsPEG-SG4 and PEG-SH4; and the components are mixed through the mixingconnector by repeatedly transferring the contents from one syringe tothe other. After complete mixing, the entire mixture iss pushed into oneof the syringes, which is then attached to one inlet of an applicator(MICROMEDICS® air assisted spray-applicator (Model SA-6105)). Syringe 3containing the pH 9.7 solution is attached onto the other inlet of theapplicator. The formulation is applied to a tissue surface as specifiedby the applicator manufacturer.

Example 19 MPA Loaded Microspheres in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg) and 10% PTXloaded MePEG5000-PDLLA (65:35) microspheres (0.5 or 2 mg) (preparedusing the procedure described in Example 3 and 4). A 1 mL capped syringe(syringe 2) is filled with 0.25 mL of 6.3 mM HCl solution (pH 2.1). A 1mL capped syringe (syringe 3) is filled 0.25 mL 0.12 M monobasic sodiumphosphate and 0.2 M sodium carbonate (pH 9.7) buffer. The components aremixed and applied to a tissue surface using the procedure described inExample 12.

Example 20 Preparation of Silk Powder(Hydrolysis)

Several pieces of silk braid (Ethicon 4-0, 638) are cut into lengths ofapprox 0.4 cm. These cut pieces are placed in a 100 ml round bottomflask that contains 50 ml 2M NaOH. The sample is stirred using amagnetic stirrer at room temperature for 24 h. The sample is neutralizedusing concentrated HCl. The neutralized contents are then dialyzedagainst deionized water using Spectrum cellulose-based dialysis tubing(WMCO approx 3000). The sample is dialyzed for 48 hours with 5 waterchanges. The dialyzed sample is then poured into a 100 ml round bottomflask. The sample is frozen and freeze-dried to yield a fluffy powderedmaterial.

Example 21 Preparation of Silk Powder Using a Cryomill

Fibers of degummed silk were cut into pieces approximately 1-2 cm inlength. The material was then milled to a powder using a cryomill (SpexCertiprep Freezer/Mill—Model 6850). A portion of the milled powder wasthen sieved through a series of different sized metal sieves to obtainsilk powder of different size ranges.

Example 22 Silk in a Multifunctional Compound

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (100 mg) and 10% silkpowder (Example 34 and Example 35). A 1 mL capped syringe (syringe 2) isfilled with 0.25 mL of 6.3 mM HCl solution (pH 2.1). A 1 mL cappedsyringe (syringe 3) is filled 0.25 mL 0.12 M monobasic sodium phosphateand 0.2 M sodium carbonate (pH 9.7) buffer. The components are mixed andapplied to a tissue surface using the procedure described in Example 12.

Example 23 In Vivo Evaluation of Perivascular Silk Powder to AssessScarring

A rat carotid artery model is described for determining whether asubstance stimulates fibrosis. Wistar rats weighing 300 g to 400 g areanesthetized with halothane. The skin over the neck region is shaved andthe skin is sterilized. A vertical incision is made over the trachea andthe left carotid artery is exposed. Silk powder formulation (Example 36and Example 46) is applied on the exposed artery that is then wrappedwith a PU film. Carotids wrapped with PU films only are used as acontrol group. The wound is closed and the animal is recovered. After 28days, the rats are sacrificed with carbon dioxide and pressure-perfusedat 100 mmHg with 10% buffered formaldehyde. Both carotid arteries areharvested and processed for histology. Serial cross-sections will be cutevery 2 mm in the treated left carotid artery and at correspondinglevels in the untreated right carotid artery. Sections are stained withH&E and Movat's stains to evaluate tissue growth around the carotidartery. Area of tunica intima, tunica media, and perivasculargranulation tissue is quantified by computer-assisted morphometricanalysis.

Example 24 Mycophenolic Acid in a Multifunctional Compound/PremixComposition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg), and MPA (60 mg, sifted <100micron). A 1 mL capped syringe (syringe 2) is filled with 0.25 mL of 6.3mM HCl solution (pH 2.1). A 1 mL capped syringe (syringe 3) is filledwith 0.35 mL 0.24 M monobasic sodium phosphate and 0.4 M sodiumcarbonate (pH 10.0) buffer. The solid contents of syringe 1 and theacidic solution of syringe 2 are mixed through a mixing connector byrepeatedly transferring the contents from one syringe to the other.After complete mixing, the entire mixture is pushed into one of thesyringes. The syringe containing the mixture then is attached to oneinlet of an applicator (MICROMEDICS® air assisted spray-applicator(Model SA-6105)). Syringe 3 containing the pH 9.7 solution is attachedonto the other inlet of the applicator. The formulation is applied to atissue surface as specified by the applicator manufacturer.

Example 25 Mycophenolic Acid and Disodium Salt of MPA (Na₂MPA) in aMultifunctional Compound/Premix Composition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg). A 1 mL capped syringe (syringe 2)is filled with 0.25 mL of 6.3 mM HCl solution (pH 2.1). A 1 mL cappedsyringe (syringe 3) is filled with 0.25 mL 0.12 M monobasic sodiumphosphate and 0.2 M sodium carbonate (pH 9.7) buffer. A 1 mL syringe(syringe 4) equipped with luer-lock mixing connector is filled with MPA(5 mg) and Na₂MPA (95 mg), both sifted <100 micron. The contents ofsyringe 4 and syringe 2 are mixed through a mixing connector byrepeatedly transferring the contents from one syringe to the other. Thissolution is then used to reconstitute the solids in syringe 1. Aftercomplete mixing, all of the formulation is pushed into one of thesyringes, which is then attached to one inlet of an applicator(MICROMEDICS® air assisted spray-applicator (Model SA-6105)). Syringe 3containing the pH 9.7 solution is attached onto the other inlet of theapplicator. The formulation is applied to a tissue surface as specifiedby the applicator manufacturer.

Example 26 Chlorpromazine in a Multifunctional Compound/PremixComposition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg), and CPZ (5 or 10 mg). A 1 mL cappedsyringe (syringe 2) is filled with 0.25 mL of 6.3 mM HCl solution (pH2.1). A 1 mL capped syringe (syringe 3) is filled 0.25 mL 0.12 Mmonobasic sodium phosphate and 0.2 M sodium carbonate (pH 9.7) buffer.The components are mixed and applied to a tissue surface using theprocedure described in Example 12.

Example 27 Paclitaxel-Loaded Microspheres in a MultifunctionalCompound/Premix Composition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg), and 10% PTX loaded MePEG5000-PDLLA(65:35) microspheres prepared by spray drying (0.5 or 2 mg) (preparedusing the procedure described in Example 3). A 1 mL capped syringe(syringe 2) is filled with 0.25 mL of 6.3 mM HCl solution (pH 2.1). A 1mL capped syringe (syringe 3) is filled 0.25 mL 0.12 M monobasic sodiumphosphate and 0.2 M sodium carbonate (pH 9.7) buffer. The components aremixed and applied to a tissue surface using the procedure described inExample 12.

Example 28 CPZ-Loaded Microspheres in a Multifunctional Compound/PremixComposition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg), and 10% CPZ loaded MePEG5000-PDLLA(65:35) microspheres prepared by spray drying (50 or 100 mg) (preparedusing the procedure described in Example 3). A 1 mL capped syringe(syringe 2) is filled with 0.25 mL of 6.3 mM HCl solution (pH 2.1). A 1mL capped syringe (syringe 3) is filled 0.25 mL 0.12 M monobasic sodiumphosphate and 0.2 M sodium carbonate (pH 9.7) buffer. The components aremixed and applied to a tissue surface using the procedure described inExample 12.

Example 29 MPA-Loaded Microspheres in Multifunctional Compound/PremixComposition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg), and 10% MPA loaded MePEG5000-PDLLA65:35 microspheres prepared by spray drying (25 or 75 mg) (preparedusing the procedure described in Example 3). A 1 mL capped syringe(syringe 2) is filled with 0.25 mL 6.3 mM HCl solution (pH 2.1). A 1 mLcapped syringe (syringe 3) iss filled 0.35 mL 0.24 M monobasic sodiumphosphate and 0.4 M sodium carbonate (pH 10.0) buffer. The componentsare mixed and applied to a tissue surface using the procedure describedin Example 12.

Example 30 Incorporation of Ptx Loaded Micelles into a MultifunctionalCompound/Premix Composition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg). A 2 mL serum vial is filled with1.5 mL of 6.3 mM HCl solution (pH 2.1). A 1 mL capped syringe (syringe2) is filled with 0.25 mL 0.12 M monobasic sodium phosphate and 0.2 Msodium carbonate (pH 9.7) buffer. A 2 mL serum vial is filled with 10%PTX loaded micelles (2 mg or 8 mg) (prepared as in Example 7) andreconstituted with 1 mL of the pH 2.1 solution. 0.25 mL of the micellesolution iss removed with a 1 mL syringe; the syringe iss attached tosyringe 1 containing the solids PEG-SG4 and PEG-SH4; and the componentsare mixed through the mixing connector by repeatedly transferring thecontents from one syringe to the other. After complete mixing, theentire mixture iss pushed into one of the syringes, which is thenattached to one inlet of an applicator (MICROMEDICS® air assistedspray-applicator (Model SA-6105)). Syringe 3 containing the pH 9.7solution is attached onto the other inlet of the applicator. Theformulation iss applied to a tissue surface as specified by theapplicator manufacturer.

Example 31 Silk in a Multifunctional Compound/Premix Composition

A 1 mL syringe (syringe 1) equipped with a luer-lock mixing connector isfilled with a multifunctional compound (Example 1) (50 mg), a mixture ofPEG-SG4 (25 mg) and PEG-SH4 (25 mg) and 10% silk powder (Example 20 andExample 21). A 1 mL capped syringe (syringe 2) is filled with 0.25 mL of6.3 mM HCl solution (pH 2.1). A 1 mL capped syringe (syringe 3) isfilled 0.25 mL 0.12 M monobasic sodium phosphate and 0.2 M sodiumcarbonate (pH 9.7) buffer. The components are mixed and applied to atissue surface using the procedure described in Example 12.

We claim:
 1. A kit for use in medical applications, comprising: a. aplurality of multifunctional compounds, each compound having thestructure of formula (I):

wherein: n is an integer from 1-12, and when n is 2-12, each Z componentmay be different; R is selected from hydrophilic polymers, hydrophobicpolymers, amphiphilic polymers, C₂₋₁₄ hydrocarbyls, andheteroatom-containing C₂₋₁₄ hydrocarbyls; X, Y, and Z are reactivegroups and can be the same or different, wherein the reactive groups areselected from nucleophilic groups, electrophilic groups, redox groups,oxidative coupling reactive groups, photoinitiated reactive groups, andtemperature-sensitive groups; L¹, L², and L³ are linking groups; and p,q, and r are integers from 0-1; wherein each reactive group is capableof reacting with at least one other reactive group, and wherein thecompound is essentially non-reactive in an initial environment but isrendered reactive upon exposure to a modification in the initialenvironment that provides a modified environment such that a pluralityof the multifunctional compounds inter-react in the modified environmentto form a three-dimensional matrix; b. a first buffer solution having apH within the range of about 1.0 to 5.5; and c. a second buffer solutionhaving a pH within the range of about 6.0 to 11.0, wherein eachcomponent is packaged separately and admixed immediately prior to use.2. A kit for use in medical applications, comprising: a. a plurality ofmultifunctional compounds, each compound having the structure of formula(I):

wherein: n is an integer from 1-12, and when n is 2-12, each Z componentmay be different; R is selected from hydrophilic polymers, hydrophobicpolymers, amphiphilic polymers, C₂-₁₄ hydrocarbyls, andheteroatom-containing C₂₋₁₄ hydrocarbyls; X, Y, and Z are reactivegroups and can be the same or different, wherein the reactive groups areselected from nucleophilic groups, electrophilic groups, redox groups,oxidative coupling reactive groups, photoinitiated reactive groups, andtemperature-sensitive groups; L¹, L², and L³ are linking groups; and p,q, and r are integers from 0-1; wherein each reactive group is capableof reacting with at least one other reactive group, and wherein thecompound is essentially non-reactive in an initial environment but isrendered reactive upon exposure to a modification in the initialenvironment that provides a modified environment such that a pluralityof the multifunctional compounds inter-react in the modified environmentto form a three-dimensional matrix; b. a first buffer solution having apH within the range of about 1.0 to 5.5; and c. a second buffer solutionhaving a pH within the range of about 6.0to 11.0, wherein each componentis packaged separately and admixed immediately prior to use, and whereinthe nucleophilic groups are amino groups and the electrophilic groupsare amine-reactive groups.
 3. The kit of claim 2, wherein theamine-reactive groups are selected from carboxylic acid esters, acidchloride groups, anhydrides, ketones, aldehydes, halo, isocyanato,thioisocyanato, epoxides, activated hydroxyl groups, olefins, carboxyl,succinimidyl ester, sulfosuccinimidyl ester, maleimido, epoxy, andethenesulfonyl.
 4. The kit of claim 1, wherein the nucleophilic groupsare sulfhydryl groups and the electrophilic groups aresulfhydryl-reactive groups are selected so as to form a thioester,imido-thioester, thioether, or disulfide linkage upon reaction with thesulfhydryl groups.
 5. The kit of claim 4 wherein the sulfhydryl-reactivegroups form a disulfide linkage and have the structure —S—S—Ar where Aris a substituted or unsubstituted nitrogen-containing heteroaromaticmoiety or a non-heterocyclic aromatic group substituted with anelectron- withdrawing moiety.
 6. The kit of claim 4 wherein thesulfhydryl-reactive groups form a thioether linkage and are selectedfrom maleimido, substituted maleimido, haloalkyl, epoxy, imino,aziridino, olefins, and α,β-unsaturated aldehydes and ketones.
 7. A kitfor use in medical applications, comprising: d. a plurality ofmultifunctional compounds, each compound having the structure of formula(I):

wherein: n is an integer from 1-12, and when n is 2-12, each Z componentmay be different; R is selected from hydrophilic polymers, hydrophobicpolymers, amphiphilic polymers, C₂-₁₄ hydrocarbyls, andheteroatom-containing C₂₋₁₄ hydrocarbyls; X, Y, and Z are reactivegroups and can be the same or different, wherein the reactive groups areselected from nucleophilic groups, electrophilic groups, redox groups,oxidative coupling reactive groups, photoinitiated reactive groups, andtemperature-sensitive groups; L¹, L², and L³ are linking groups; and p,q, and r are integers from 0-1; wherein each reactive group is capableof reacting with at least one other reactive group, and wherein thecompound is essentially non-reactive in an initial environment but isrendered reactive upon exposure to a modification in the initialenvironment that provides a modified environment such that a pluralityof the multifunctional compounds inter-react in the modified environmentto form a three-dimensional matrix; e. a first buffer solution having apH within the range of about 1.0 to 5.5; and f. a second buffer solutionhaving a pH within the range of about 6.0 to 11.0, wherein eachcomponent is packaged separately and admixed immediately prior to use,and wherein the reactive groups are selected from reactive groups thata) undergo an oxidation-reduction reaction and are vinyl groups, b) areoxidative coupling reactive groups and are halo groups, with an adjacentelectron-withdrawing group on the halogen-bearing carbon, c) arephotoinitiated reactive groups and are selected from azide, alkyl, andbenzophenone, and d) are temperature sensitive groups and are vinylgroups.
 8. The kit of claim 1, wherein R is a hydrophilic polymer. 9.The kit of claim 8, wherein the hydrophilic polymer is a linear,branched, dendrimeric, hyperbranched, or star polymer.
 10. The kit ofclaim 9, wherein the hydrophilic polymer is selected from polyalkyleneoxides; polyols; poly(oxyalkylene)-substituted diols and polyols;polyoxyethylated sorbitol; polyoxyethylated glucose; poly(acrylic acids)and analogs and copolymers thereof; polymaleic acids; polyacrylamides;poly(olefmic alcohols); poly(N-vinyl lactams); polyoxazolines;polyvinylamines; and copolymers thereof.
 11. The kit of claim 10,wherein the hydrophilic polymer is a poly(oxyalkylene)-substitutedpolyol selected from mono-, di- and tri-polyoxyethylated glycerol, mono-and di-polyoxyethylated propylene glycol, and mono- and di-polyoxyethylated trimethylene glycol, andtetra(oxyethylene)pentaerythritol.
 12. The kit of claim 1, wherein priorto use, each component is in a separate sterile package.
 13. The kit ofclaim 1, further comprising a delivery device.
 14. The kit of claim 13,wherein the delivery device is a multi-compartment device.
 15. The kitof claim 14, wherein the multi-component device is amultiple-compartment syringe system having multiple barrels, a mixinghead, and an exit orifice.
 16. The kit of claim 15, wherein themultifunctional compound, the first buffer solution, and the secondbuffer solution are housed separately in the multiple-component syringesystem.